Dock 3 tumor suppressor gene

ABSTRACT

The invention relates to a newly identified tumor suppressor gene, designated DOS (for Deleted in Osteosarcoma and alternatively referred to herein as DOCK 3) which has been cloned from human and mouse cells. The DOS nucleic acid and protein molecules and their use in the diagnosing and treating disorders characterized by aberrant DOS molecule expression are described.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to U.S.provisional application Ser. No. 60/297,382, filed Jun. 11, 2001.

GOVERNMENT SUPPORT

This invention was made in part with government support under grantnumber R01CA58596 and 5T32DK07191-26 from the NIH. The government mayhave certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to a novel tumor suppressor gene that has beencloned from human and mouse tissue. The invention is directed to theisolated tumor suppressor nucleic acid, the proteins encoded by thesenucleic acids, binding agents that selectively bind thereto, and variousdiagnostic, therapeutic and research uses of these compositions.

BACKGROUND OF THE INVENTION

Cancer progression is caused by accumulation of multiple mutations thatprovide selective advantage during cancer growth, invasion andmetastasis (1, 2, 3). While gain of function mutations occur inoncogenes, many of the genetic events that underlie cancer appear to beinactivating, or loss of function mutations affecting tumor suppressorgenes (1). Tumor suppressor gene identified to date exhibit diversecellular functions (4). Functional studies on these tumor suppressorgenes have supported the original hypothesis that these genes representpotential bottlenecks in wide variety of cellular pathways (4). Theseinclude proliferation, differentiation, apoptosis and response to DNAdamage. For example p53 and WT1 are DNA binding transcription factors;RB1, APC and possibly BRCA1 indirectly modulate transcription; P16 is aninhibitor of kinases required for cell cycle progression; PTEN is anovel phosphatase; NF2 is a cell structural component; VHL is apotential mediator of mRNA processing. A large number of genes arebelieved to be genomic caretakers and mutations in these genes causemicrosatellite instability (MSH2, MLH1, PMS1 and PMS2) or chromosomalinstability (p53, possibly BRCA1 and BRCA2). To date, genes involved inadvanced stages of cancer progression such as invasion, angiogenesis andmetastasis have not been identified (4, 5). They are likely to becomeevident over time with large-scale genome wide analysis.

The vast majority of cancers result from sporadic genetic events andonly rare cases (less than 1%) have an inherited component (7, 8).However, the isolation of tumor suppressor genes has typicallyoriginated from genetic analysis of such rare inherited cancer syndromes(7). Linkage analysis on large families with cancer present in multiplegenerations allows identification of markers that co-segregate withcancer. In some cases cytogenetic abnormalities could also be observedeither in sporadic or in germline tumors (7,9). For example, a smallfraction of retinoblastomas have a homozygous deletion of RB1 gene (7).Rare Wilms tumor and colon cancer have deletions of WT1 and APC,respectively. These germline or sporadic homozygous deletions have beeninstrumental in tumor suppressor gene cloning efforts (7).

Allelic losses in tumors are typically detected as “loss ofheterozygosity” or “LOH”. This represents loss of a polymorphic marker,commonly resulting from a large interstitial deletion or chromosomalnon-disjunction event. While LOH is a common event in cancer, it onlyallows rough mapping of tumor suppressor loci (9). The large size of theLOH region (>10 Mb) makes the identification of the specific tumorsuppressor gene targeted by mutation difficult. In contrast, homozygousdeletions in tumors are typically small (<100 Kb) since they arerestricted by the deletion of the flanking genes. Homozygous deletionsoccur by diverse mechanisms, including a small deletion in one alleleaccompanied by LOH of the second allele, or even large deletion of eachallele whose common region of overlap is small. Identification of suchhomozygous deletions can be a powerful approach to identify tumorsuppressor genes (7).

Significant technological advances have been made to identify regions ofchromosomes involved in tumor progression. Analyses of metaphasechromosomes show chromosomal rearrangements in leukemia and lymphomas(10). This is more difficult in solid tumors where karyotyping is lesscommonly performed. Fluorescence in situ hybridization (FISH) hasgreatly improved the sensitivity and specificity of detecting chromosomeaberrations (11, 12). However, its application in human malignancies isstill limited because of complex karyotypes seen in clinical samples.Comparative genome hybridization (CGH) uses both normal and tumorgenomes to identify regions in tumor DNA that have undergone changes incopy number (13). In this technique, normal and tumor DNA are labeledwith two different haptens that fluoresce at different wavelengths. Theprobes are then hybridized to metaphase chromosomes in the presence ofexcess Cot-1 DNA thus inhibiting hybridization of labeled repetitivesequences. The ratio of the amount of two genomes that hybridize tospecific areas of the chromosomes indicates the copy number of the twosamples. CGH is currently limited to a resolution of 10 to 20 Mb andmore sensitive in detecting amplifications rather than a small deletion(9). An alternative method, Representational Difference Analysis (RDA)is a PCR based subtractive hybridization technique, that is particularlyapplicable in isolating homozygous deletions in tumors (14, 18, 19). Ithas already been successful in isolating tumor suppressor genes PTEN andDMBT1 and has played a significant role in cloning of BRCA2 (15, 16,17).

In view of the foregoing, a need exists to identify novel tumorsuppressor genes to detect and treat various cancers. Preferably, suchsuppressor genes will have unique sequences that will permit thetargeting of therapeutic agents for treating such cancers and thedevelopment of agents for detecting such cancers.

SUMMARY OF THE INVENTION

The invention provides novel human and mouse tumor suppressor genes andis based, in part, on the discovery that this novel gene is deleted in amouse osteosarcoma cell line. Using Representational Difference Analysis(RDA) on a mouse tumor model, we found a region of homozygous deletionin the mouse cell line. This region of deletion is homologous to humanchromosome 7q31. We have cloned the gene residing in the deleted segment(DOS, for deleted in osteosarcoma, alternatively referred to herein asDOCK 3) from both mouse and humans. Human and mouse DOS protein sequenceis about 97% identical. The protein has limited homology (approximately30% identity) with three known genes, namely, DOCK180, myoblast city,and Ced 5. These three proteins are evolutionary conserved in bothsequence and function and regulate actin cytoskeleton during cellmigration. Accordingly, although not wishing to be bound to anyparticular theory or mechanism, we believe the DOS gene plays a role inregulating actin cytoskeleton in cell growth and cancer. Thus, theinvention is directed to novel compositions of the DOS nucleic acids andproteins encoded thereby, as well as to agents that selectively bind tothese novel molecules, and to diagnostic, therapeutic, and researchapplications of these compositions.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule is selectedfrom the group consisting of:

(a) nucleic acid molecules which hybridize under stringent conditions toa nucleic acid molecule having a nucleotide sequence set forth as SEQ IDNO:1 or SEQ ID NO:3, and which code for a DOS protein,

(b) deletions, additions and substitutions of the nucleic acid moleculesof (a),

(c) nucleic acid molecules that differ from the nucleic acid moleculesof (a) or (b) in codon sequence due to the degeneracy of the geneticcode, and

(d) complements of (a), (b) or (c).

The preferred isolated nucleic acids of the invention are DOS nucleicacid molecules which encode a DOS protein. As used herein, a DOS proteinrefers to a protein which is encoded by a nucleic acid having SEQ IDNO:1 or SEQ ID NO:3, or a functional fragment thereof, provided that thefunctional fragment encodes a protein which plays a role in tumorsuppression, cytoskeletal organization, cell proliferation, cellmigration, cellular growth and development, and/or cell-cellinteraction.

In the preferred embodiments, the isolated nucleic acid molecule is SEQID NO:1 or SEQ ID NO:3

According to another aspect of the invention, further isolated nucleicacid molecules that are based on the above-noted DOS nucleic acidmolecules are provided. In this aspect, the isolated nucleic acidmolecules are selected from the group consisting of:

(a) a unique fragment of the nucleotide sequence set forth as SEQ IDNO:1 or set forth as SEQ ID NO:3 between 12 and 115 nucleotides inlength or more and

(b) complements of (a),

wherein the unique fragments exclude nucleic acids having nucleotidesequences that are contained within SEQ ID NO:1 or SEQ ID NO:3, and thatare known as of the filing date of the priority application.

In one embodiment of the invention an isolated unique nucleic acidfragment comprising SEQ ID NO: 31 of SEQ ID NO: 1 is provided.

In yet another aspect of the invention, Mutant DOS nucleic acidmolecules are provided. The Mutant DOS nucleic acid molecules contain asequence which is identical to SEQ ID NO:1 or SEQ ID NO:3, with theexception that the sequence includes one or more mutations, e.g., pointmutations, deletion mutations, such that the Mutant DOS nucleic acidmolecule does not encode a functional DOS protein. Rather, the MutantDOS nucleic acid molecules encode a Mutant DOS protein, i.e., a proteinwhich does not exhibit a DOS protein functional activity.

In preferred embodiments, the binding polypeptide is an antibody orantibody fragment, more preferably, an Fab or F(ab)₂ fragment of anantibody. Typically, the fragment includes a CDR3 region that isselective for the DOS protein or Mutant DOS protein. Any of the varioustypes of antibodies can be used for this purpose, including monoclonalantibodies, humanized antibodies and chimeric antibodies.

According to a further aspect of the invention, pharmaceuticalcompositions containing the nucleic acids, proteins, and bindingpolypeptides of the invention are provided. The pharmaceuticalcompositions contain any of the foregoing therapeutic agents in apharmaceutically acceptable carrier. Thus, in a related aspect, theinvention provides a method for forming a medicament that involvesplacing a therapeutically effective amount of the therapeutic agent inthe pharmaceutically acceptable carrier to form one or more doses.

According to another aspect of the invention, various diagnostic methodsare provided. In general, the methods are for diagnosing “a disordercharacterized by aberrant expression of a DOS molecule”. As used herein,“aberrant expression” refers to either or both of a decreased expression(including no expression) of the DOS molecule (nucleic acid or protein)or an increased expression of a “Mutant DOS molecule”. A Mutant DOSmolecule refers to a DOS nucleic acid molecule which includes a mutation(point mutation, deletion) or to a DOS protein molecule (e.g., geneproduct of mutant DOS nucleic acid molecule) which includes a mutation,provided that the mutation results in a mutant DOS protein that does nothave the DOS functional activity that is exhibited by a DOS protein asdescribed herein. The diagnostic methods of the invention can be used todetect the presence of a disorder associated with aberrant expression ofa DOS molecule, as well as to assess the progression and/or regressionof the disorder such as in response to treatment (e.g., chemotherapy,radiation).

According to this aspect of the invention, the method for diagnosing adisorder characterized by aberrant expression of a DOS moleculeinvolves: detecting in a first biological sample obtained from asubject, expression of a DOS molecule or a Mutant DOS molecule; whereindecreased expression of a DOS molecule or the increased expression of aMutant DOS molecule compared to a control sample indicates that thesubject has a disorder characterized by aberrant expression of a DOSmolecule.

As used herein, a “disorder characterized by aberrant expression of aDOS molecule” refers to a disorder in which there is a detectabledifference in the expression levels of DOS molecule(s) and/or Mutant DOSmolecule(s) in selected cells of a subject compared to the

According to another aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule is selectedfrom the group consisting of:

(a) nucleic acid molecules which hybridize under stringent conditions toa nucleic acid molecule having a nucleotide sequence selected from thegroup consisting of SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, or 29,

(b) deletions, additions and substitutions of the nucleic acid moleculesof (a),

(c) nucleic acid molecules that differ from the nucleic acid moleculesof (a) or (b) in codon sequence due to the degeneracy of the geneticcode, and

(d) complements of (a), (b) or (c)

According to yet another aspect of the invention, an expression vectorcomprising any of the isolated nucleic acid molecules of the inventionoperably linked to a promoter are provided. In a related aspect, hostcells transformed or transfected with such expression vectors also areprovided.

According to still a further aspect of the invention, a transgenicnon-human animal comprising an expression vector of the invention isprovided. Also provided is a transgenic non-human animal which hasreduced expression of a DOS nucleic acid molecule or of a Mutant DOSnucleic acid molecule.

According to another aspect of the invention, an isolated polypeptideencoded by any of the foregoing isolated nucleic acid molecules of theinvention is provided. Preferably, the isolated polypeptide comprises apolypeptide sequence selected from the group, consisting of SEQ ID NO:2, 4, 9, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.

In one aspect of the invention a functional protein fragment of SEQ IDNO: 2 comprising SEQ ID NO: 32 is provided.

In yet a further aspect of the invention, binding polypeptides thatselectively bind to a DOS molecule and/or to a Mutant DOS molecule areprovided. According to this aspect, the binding polypeptides bind to anisolated nucleic acid or protein of the invention, including binding tounique fragments thereof. Preferably, the binding polypeptides bind to aDOS protein, a Mutant DOS protein, or a unique fragment thereof. Incertain particularly preferred embodiments, the binding polypeptidebinds to a DOS protein but does not bind to a Mutant DOS protein, i.e.,the binding polypeptides are selective for binding to the Mutant proteinand can be used in various assays to detect the presence of the MutantDOS protein without detecting DOS protein. control levels of thesemolecules. Thus, a disorder characterized by aberrant expression of aDOS molecule embraces underexpression (including no expression) of a DOSnucleic acid molecule or a DOS protein compared to control levels ofthese molecules, as well as overexpression of a Mutant DOS nucleic acidmolecule or Mutant DOS protein compared to control levels of thesemolecules. Such differences in expression levels can be determined inaccordance with the diagnostic methods of the invention as disclosedherein. Exemplary disorders that are characterized by aberrantexpression of a DOS molecule include: various cancers and disordersassociated with abnormal cytoskeleton organization, cell proliferation,cell migration, cellular growth and development, and/or cell-cellinteraction.

In certain embodiments, the methods of the invention are to diagnose acancer including, but not limited to, biliary tract cancer, brain cancer(including glioblastomas and medulloblastomas), breast cancer; cervicalcancer; choriocarcinoma, colon cancer, endometrial cancer, esophagealcancer, gastric cancer, hematological neoplasms, including acutelymphocytic and myelogenous leukemia, multiple myeloma, AIDS associatedleukemias and adult T-cell leukemia lymphoma, intraepithelial neoplasms,including Bowen's disease and Paget's disease, liver cancer, lungcancer, lymphomas, including Hodgkin's disease and lymphocyticlymphomas, neuroblastomas, oral cancer, including squamous cellcarcinoma, ovarian cancer, including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells, pancreaticcancer, prostate cancer, rectal cancer, renal cancer includingadenocarcinoma and Wilms tumor, sarcomas, including leiomyosarcoma,rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma, skincancer, including melanoma, Kaposi's sarcoma, basocellular cancer andsquamous cell cancer, testicular cancer, including germinal tumors(seminomas, and non-seminomas such as teratomas and choriocarcinomas),stromal tumors and germ cell tumors, and thyroid cancer, includingthyroid adenocarcinoma and medullary carcinoma. In the preferredembodiments, the methods of the invention are useful for diagnosingWilms tumor, ovarian carcinoma, renal cell carcinoma, osteosarcomafibrosarcoma, prostate cnacer, colon cancer, and brain cancer.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the progression of a disorder. According to theseembodiments, the methods further involve: detecting in a secondbiological sample obtained from the subject, expression of a DOSmolecule or a Mutant DOS molecule, and comparing the expression of theDOS molecule or the Mutant DOS molecule in the first biological sampleand the second biological sample. In these embodiments, a decrease inthe expression of the DOS molecule in the second biological samplecompared to the first biological sample or an increase in the expressionof the Mutant DOS molecule in the second biological sample compared tothe first biological sample indicates progression of the disorder.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the regression of a disorder. According to these embodiments,the methods further involve: detecting in a second biological sampleobtained from the subject, expression of a DOS molecule or a Mutant DOSmolecule, and comparing the expression of the DOS molecule or the MutantDOS molecule in the first biological sample and the second biologicalsample. In these embodiments, an increase in the expression of the DOSmolecule in the second biological sample compared to the firstbiological sample or a decrease in the expression of the Mutant DOSmolecule in the second biological sample compared to the firstbiological sample indicates regression of the disorder.

In certain embodiments, the diagnostic methods of the invention detect aDOS molecule that is a DOS nucleic acid molecule or a Mutant DOS nucleicacid molecule as described above. In yet other embodiments, the methodsinvolve detecting a DOS protein or Mutant DOS protein as describedabove.

Various detection methods can be used to practice the diagnostic methodsof the invention. For example, when the methods can involve contactingthe biological sample with an agent that selectively binds to the DOSmolecule or to the Mutant DOS molecule to detect these molecules. Incertain embodiments, the DOS molecule is a nucleic acid and the methodinvolves using an agent that selectively binds to the DOS molecule or tothe Mutant DOS molecule, e.g., a nucleic acid that hybridizes understringent conditions to a nucleic acid molecule selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, and 29. In yet other embodiments, the DOS molecule is a protein andthe method involves using an agent that selectively binds to the DOSmolecule or to the Mutant DOS molecule, e.g., a binding polypeptide,such as an antibody, that selectively binds to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, and 30.

In yet another embodiment an agent that selectively binds a fragment ofa DOS molecule or to a fragment of a Mutant DOS molecule is provided. Incertain embodiments the fragment of the DOS molecule or the fragment ofthe DOS molecule or the fragment of the Mutant DOS molecule is a nucleicacid molecule. One preferred nucleic acid fragment of the DOS moleculecompressers SEQ ID NO: 31. In other embodiments, the fragment of the DOSmolecule or the fragment of the Mutant DOS molecule is a polypeptide.One preferred polypeptide of the DOS molecule comprises SEQ ID NO: 32.

According to still another aspect of the invention, kits for performingthe diagnostic methods of the invention are provided. The kits includenucleic acid-based kits or protein-based kits. According to the formerembodiment, the kits include: one or more nucleic acid molecules thathybridize to a DOS nucleic acid molecule or to a Mutant DOS nucleic acidmolecule under stringent conditions; one or more control agents; andinstructions for the use of the nucleic acid molecules, and agents inthe diagnosis of a DOS tumor. As used herein, A DOS tumor is disorderassociated with aberrant expression of a DOS molecule. Nucleicacid-based kits optionally further include a first primer and a secondprimer, wherein the first primer and the second primer are constructedand arranged to selectively amplify at least a portion of an isolatedDOS nucleic acid molecule selected from the group consisting of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29.Alternatively, protein based-kits are provided. Such kits include: oneor more binding polypeptides that selectively bind to a DOS protein or aMutant DOS protein; one or more control agents; and instructions for theuse of the binding polypeptides, and agents in the diagnosis of adisorder associated with aberrant expression of a DOS molecule. In thepreferred embodiments, the binding polypeptides are antibodies orantigen-binding fragments thereof, such as those described above. Inthese and other embodiments, certain of the binding polypeptides bind tothe Mutant DOS protein but do not bind to the DOS protein to furtherdistinguish the expression of these proteins in a biological sample.

The invention also provides treatment methods. In general, the treatmentmethods involve administering an agent to increase expression of a DOSmolecule and/or reduce expression of a Mutant DOS molecule. Thus, thesemethods include gene therapy applications. In certain embodiments, themethod for treating a subject with a disorder characterized by aberrantexpression of a DOS molecule, involves administering to the subject aneffective amount of a DOS nucleic acid molecule to treat the disorder.In yet other embodiments, the method for treatment involvesadministering to the subject an effective amount of an anti-sensemolecule to inhibit (reduce/eliminate) expression of a Mutant DOSnucleic acid molecule and, thereby, treat the disorder. An exemplarymolecule for inhibiting expression of a Mutant DOS nucleic acid moleculeis an anti-sense molecule that is selective for the mutant nucleic acidand that does not inhibit expression of the DOS nucleic acid molecule.Alternatively, the method for treating a subject with a disordercharacterized by aberrant expression of a DOS molecule involvesadministering to the subject an effective amount of a DOS protein totreat the disorder. In yet another embodiment, the treatment methodinvolves administering to the subject an effective amount of a bindingpolypeptide to inhibit a Mutant DOS protein and, thereby, treat thedisorder. In certain preferred embodiments, the binding polypeptide isan antibody or an antigen-binding fragment thereof; more preferably, theantibodies or antigen-binding fragments are labeled with one or morecytotoxic agents

The invention provides various research methods and compositions. Thus,according to one aspect of the invention, a method for producing a DOSprotein is provided. The method involves providing a DOS nucleic acidmolecule operably linked to a promoter, wherein the DOS nucleic acidmolecule encodes the DOS protein or a fragment thereof; expressing theDOS nucleic acid molecule in an expression system; and isolating the DOSprotein or a fragment thereof from the expression system. Preferably,the DOS nucleic acid molecule has SEQ ID NO:1 or SEQ ID NO:3. Accordingto yet another aspect of the invention, a method for producing a MutantDOS protein is provided. This method involves: providing a Mutant DOSnucleic acid molecule operably linked to a promoter, wherein the MutantDOS nucleic acid molecule encodes the Mutant DOS protein or a fragmentthereof; expressing the Mutant DOS nucleic acid molecule in anexpression system; and isolating the Mutant DOS protein or a fragmentthereof from the expression system. Preferably, the Mutant DOS nucleicacid molecule has SEQ ID NO:1 or SEQ ID NO:3, with one or more pointmutations or deletions to encode a Mutant DOS protein.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention in one aspect involves the cloning of a cDNAencoding a DOS protein. The sequence of the human gene is presented asSEQ ID NO:1, and the predicted amino acid sequence of this gene'sprotein product is presented as SEQ ID NO:2. The sequence of the mousegene is presented as SEQ ID NO:3, and the predicted amino acid sequenceof this gene's protein product is presented as SEQ ID NO:4. Sequenceanalysis shows that the human and mouse DOS proteins are about 97%identical. The invention thus involves in one aspect DOS proteins, genesencoding those proteins, functional modifications and variants of theforegoing, useful fragments of the foregoing, as well as therapeutic anddiagnostic products and methods relating thereto.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule is selectedfrom the group consisting of:

(a) nucleic acid molecules which hybridize under stringent conditions toa nucleic acid molecule having a nucleotide sequence set forth as SEQ IDNO:1 or SEQ ID NO:3, and which code for a DOS protein,

(b) deletions, additions and substitutions of the nucleic acid moleculesof (a),

(c) nucleic acid molecules that differ from the nucleic acid moleculesof (a) or (b) in codon sequence due to the degeneracy of the geneticcode, and

(d) complements of (a), (b), or (c).

The preferred isolated nucleic acids of the invention are DOS nucleicacid molecules which encode a DOS protein. As used herein, a DOS proteinrefers to a protein which is encoded by a nucleic acid having SEQ IDNO:1 or SEQ ID NO:3, or a functional fragment thereof, or a functionalequivalent thereof (e.g., a nucleic acid sequence encoding the sameprotein as encoded by SEQ ID NO:1 or SEQ ID NO:3), provided that thefunctional fragment or equivalent encodes a protein which exhibits a DOSfunctional activity. As used herein, a DOS functional activity refers tothe ability of a DOS protein to modulate one or more of the followingparameters: cytoskeletal organization, cell growth, cell proliferation,cell migration, and/or cell-cell interactions. An exemplary DOSfunctional activity is a tumor suppressor activity such as suppressingand/or reducing tumor cell growth, proliferation, and/or metastasis.Although not wishing to be bound to any particular theory or mechanism,it is believed that the DOS protein may affect at least some of theabove-noted cell functions by interacting with actin and, thereby,modulating cytoskeletal organization.

In the preferred embodiments, the isolated nucleic acid molecule is SEQID NO:1 or SEQ ID NO:3.

The invention provides nucleic acid molecules which code for DOSproteins and which hybridize under stringent conditions to a nucleicacid molecule consisting of the nucleotide set forth in SEQ ID NO:1 orSEQ ID NO:3. Such nucleic acids may be DNA, RNA, composed of mixeddeoxyribonucleotides and ribonucleotides, or may also incorporatesynthetic non-natural nucleotides. Various methods for determining theexpression of a nucleic acid and/or a polypeptide in normal and tumorcells are known to those of skill in the art and are described furtherbelow and in the Examples. As used herein, the term protein is meant toinclude large molecular weight proteins and peptides and low molecularweight peptides or fragments thereof.

The term “stringent conditions” as used herein refers to parameters withwhich the art is familiar. Nucleic acid hybridization parameters may befound in references which compile such methods, e.g. Molecular Cloning:A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, stringentconditions, as used herein, refers, for example, to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH2PO4 (pH 7), 0.5%SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraceticacid. After hybridization, the membrane upon which the DNA istransferred is washed at 2×SSC at room temperature and then at0.1×SSC/0.1×SDS at temperatures up to 68° C.

The foregoing set of hybridization conditions is but one example ofstringent hybridization conditions known to one of ordinary skill in theart. There are other conditions, reagents, and so forth which can beused, which result in a stringent hybridization. The skilled artisanwill be familiar with such conditions, and thus they are not given here.It will be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of DOS nucleic acid molecules of the invention.The skilled artisan also is familiar with the methodology for screeningcells and libraries for expression of such molecules which then areroutinely isolated, followed by isolation of the pertinent nucleic acidmolecule and sequencing.

In general homologs and alleles typically will share at least 40%nucleotide identity and/or at least 50% amino acid identity to SEQ IDNOs:1 or 3 and SEQ ID NOs:2 or 4, respectively, in some instances willshare at least 50% nucleotide identity and/or at least 65% amino acididentity and in still other instances will share at least 60% nucleotideidentity and/or at least 75% amino acid identity. Preferred homologs andalleles share nucleotide and amino acid identities with SEQ ID NO:1 orSEQ ID NO:3 and SEQ ID NO:2 or SEQ ID NO:4, respectively, and encodepolypeptides of greater than 80%, more preferably greater than 90%,still more preferably greater than 95% and most preferably greater than99% identity. The percent identity can be calculated using various,publicly available software tools developed by NCBI (Bethesda, Md.) thatcan be obtained through the internet (ftp:/ncbi.nlm.nih.gov/pub/).Exemplary tools include the BLAST system available athttp://www.ncbi.nlm.nih.gov, which uses algorithms developed by Altschulet al. (Nucleic Acids Res. 25:3389-3402, 1997). Pairwise and ClustalWalignments (BLOSUM30 matrix setting) as well as Kyte-Doolittlehydropathic analysis can be obtained using the MacVector sequenceanalysis software (Oxford Molecular Group). Watson-Crick complements ofthe foregoing nucleic acid molecules also are embraced by the invention.

In screening for DOS proteins, a Southern blot may be performed usingthe foregoing conditions, together with a radioactive probe. Afterwashing the membrane to which the DNA is finally transferred, themembrane can be placed against X-ray film to detect the radioactivesignal.

The invention also includes degenerate nucleic acid molecules whichinclude alternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating DOS protein.Similarly, nucleotide sequence triplets which encode other amino acidresidues include, but are not limited to: CCA, CCC, CCG and CCT (prolinecodons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC,ACG and ACT (threonine codons); AAC and AAT (asparagine codons); andATA, ATC and ATT (isoleucine codons). Other amino acid residues may beencoded similarly by multiple nucleotide sequences. Thus, the inventionembraces degenerate nucleic acids that differ from the biologicallyisolated nucleic acids in codon sequence due to the degeneracy of thegenetic code.

According to another aspect of the invention, further isolated nucleicacid molecules that are based on the above-noted DOS nucleic acidmolecules are provided. In this aspect, the isolated nucleic acidmolecules are selected from the group consisting of:

(a) a unique fragment of the nucleotide sequence set forth as SEQ IDNO:1, set as SEQ ID NO:3 between 12 and 32 nucleotides in length or moreand

(b) complements of (a),

wherein the unique fragments exclude nucleic acids having nucleotidesequences that are contained within SEQ ID NO:1 or SEQ ID NO:3, and thatare known as of the filing date of this application.

The invention also provides isolated unique fragments of SEQ ID NOs:1 or3 or complements of SEQ ID NOs:1 or 3. In one embodiment, an isolatedunique nucleic acid fragment of SEQ ID NO:1 comprising SEQ ID NO:31 isprovided. A unique fragment is one that is a ‘signature’ for the largernucleic acid. It, for example, is long enough to assure that its precisesequence is not found in molecules outside of the DOS nucleic acidmolecules defined above. Those of ordinary skill in the art may apply nomore than routine procedures to determine if a fragment is unique withinthe human or mouse genome. Unique fragments, however, exclude fragmentscompletely composed of the nucleotide sequences that are containedwithin SEQ ID NO: 1 or SEQ ID NO: 3 and that are known as of the filingdate of this application.

Unique fragments can be used as probes in Southern blot assays toidentify such nucleic acid molecules, or can be used in amplificationassays such as those employing PCR. As known to those skilled in theart, large probes such as 200 nucleotides or more are preferred forcertain uses such as Southern blots, while smaller fragments will bepreferred for uses such as PCR. Unique fragments also can be used toproduce fusion proteins for generating antibodies or determining bindingof the polypeptide fragments, or for generating immunoassay components.Likewise, unique fragments can be employed to produce nonfused fragmentsof the DOS polypeptides useful, for example, in the preparation ofantibodies, in immunoassays. Unique fragments further can be used asantisense molecules to inhibit the expression of DOS nucleic acids andpolypeptides, particularly for therapeutic purposes as described ingreater detail below.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NO:1 and/or SEQ ID NO:3 and its complementwill require longer segments to be unique while others will require onlyshort segments, typically between 12 and 32 nucleotides or more inlength (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63 ,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115 or more), up to the entire length of the disclosedsequence. Many segments of the polynucleotide coding region orcomplements thereof that are 18 or more nucleotides in length will beunique. Those skilled in the art are well versed in methods forselecting such sequences, typically on the basis of the ability of theunique fragment to selectively distinguish the sequence of interest fromnon-DOS nucleic acid molecules. A comparison of the sequence of thefragment to those on known data bases typically is all that isnecessary, although in vitro confirmatory hybridization and sequencinganalysis may be performed.

A unique fragment can be a functional fragment. A functional fragment ofa nucleic acid molecule of the invention is a fragment which retainssome functional property of the larger nucleic acid molecule, such ascoding for a functional polypeptide, binding to proteins, regulatingtranscription of operably linked nucleic acid molecules, and the like.One of ordinary skill in the art can readily determine using the assaysdescribed herein and those well known in the art to determine whether afragment is a functional fragment of a nucleic acid molecule using nomore than routine experimentation.

In yet another aspect of the invention, Mutant DOS nucleic acidmolecules are provided. The Mutant DOS nucleic acid molecules contain asequence which is identical to SEQ ID NO:1 or SEQ ID NO:3, with theexception that the sequence includes one or more mutations, e.g.,deletions, additions or substitutions, such that the Mutant DOS nucleicacid molecule does not encode a functional DOS protein. Rather, theMutant DOS nucleic acid molecules encode a Mutant DOS protein, i.e., aprotein which does not exhibit a DOS protein functional activity.

According to another aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule is selectedfrom the group consisting of:

(a) nucleic acid molecules which hybridize under stringent conditions toa nucleic acid molecule having a nucleotide sequence selected from thegroup consisting of SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, or 29,

(b) deletions, additions and substitutions of the nucleic acid moleculesof (a),

(c) nucleic acid molecules that differ from the nucleic acid moleculesof (a) or (b) in codon sequence due to the degeneracy of the geneticcode, and

(d) complements of (a), (b) or (c)

As used herein with respect to nucleic acid molecules, the term“isolated” means: (i) amplified in vitro by, for example, polymerasechain reaction (PCR); (ii) recombinantly produced by cloning; (iii)purified, as by cleavage and gel separation; or (iv) synthesized by, forexample, chemical synthesis. An isolated nucleic acid molecule is onewhich is readily manipulable by recombinant DNA techniques well known inthe art. Thus, a nucleotide sequence contained in a vector in which 5′and 3′ restriction sites are known or for which polymerase chainreaction (PCR) primer sequences have been disclosed is consideredisolated but a nucleic acid sequence existing in its native state in itsnatural host is not. An isolated nucleic acid molecule may besubstantially purified, but need not be. For example, a nucleic acidmolecule that is isolated within a cloning or expression vector is notpure in that it may comprise only a tiny percentage of the material inthe cell in which it resides. Such a nucleic acid molecule is isolated,however, as the term is used herein because it is readily manipulable bystandard techniques known to those of ordinary skill in the art. Anisolated nucleic acid molecule as used herein is not a naturallyoccurring chromosome.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a Mutant DOS nucleic acid molecule encoding aMutant DOS protein. This is desirable in medical conditions wherein anaberrant DOS expression is not desirable, e.g., cancer. As used herein,a “Mutant DOS nucleic acid molecule” refers to a DOS nucleic acidmolecule which includes a mutation (addition, deletion, or substitution)such that the Mutant DOS nucleic acid molecule does not encode afunctional DOS protein. Rather, the Mutant DOS nucleic acid moleculeencodes a Mutant DOS protein, i.e., a protein which does not exhibit aDOS protein functional activity. A “Mutant DOS protein” refers to a DOSprotein that is a gene product of a mutant DOS nucleic acid moleculewhich includes a mutation that affects the functional activity of theDOS molecule. As used herein, the term “aberrant” refers to decreasedexpression (including zero or reduced expression) of the natural DOSmolecule (nucleic acid or protein) or increased expression of a MutantDOS molecule (nucleic acid or protein).

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon SEQ ID NOs:1 or 3, or upon allelic or homologousgenomic and/or cDNA sequences, one of skill in the art can easily chooseand synthesize any of a number of appropriate antisense molecules foruse in accordance with the present invention. In order to besufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 10 and, more preferably, atleast 15 consecutive bases which are complementary to the target,although in certain cases modified oligonucleotides as short as 7 basesin length have been used successfully as antisense oligonucleotides(Wagner et al., Nature Biotechnology 14: 840-844, 1996)

Most preferably, the antisense oligonucleotides comprise a complementarysequence of 20-30 bases. Although oligonucleotides may be chosen whichare antisense to any region of the gene or mRNA transcripts, inpreferred embodiments the antisense oligonucleotides correspond toN-terminal or 5′ upstream sites such as translation initiation,transcription initiation or promoter sites. In addition, 3′-untranslatedregions may be targeted. Targeting to mRNA splicing sites has also beenused in the art but may be less preferred if alternative mRNA splicingoccurs. In addition, the antisense is targeted, preferably, to sites inwhich mRNA secondary structure is not expected (see, e.g., Sainio etal., Cell Mol. Neurobiol. 14(5):439-457, 1994) and at which proteins arenot expected to bind. The present invention also provides for antisenseoligonucleotides which are complementary to genomic DNA and/or cDNAcorresponding to SEQ ID Nos: 1 and 3. Antisense to allelic or homologouscDNAs and genomic DNAs are enabled without undue experimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus, modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisense molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acid molecules encodingDOS proteins, together with pharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

According to yet another aspect of the invention, an expression vectorcomprising any of the isolated nucleic acid molecules of the invention,preferably operably linked to a promoter is provided. In a relatedaspect, host cells transformed or transfected with such expressionvectors also are provided.

Thus, it will also be recognized from the examples that the inventionembraces the use of the DOS nucleic acid molecules in expressionvectors, as well as to transfect host cells and cell lines, be theseprokaryotic (e.g., E. coli, or eukaryotic (e.g., CHO cells, COS cells,yeast expression systems and recombinant baculovirus expression ininsect cells). Especially useful are mammalian cells such as mouse,hamster, pig, goat, primate, etc. They can be of a wide variety oftissue types, including mast cells, fibroblasts, oocytes andlymphocytes, and they may be primary cells or cell lines. Specificexamples include dendritic cells, U293 cells, peripheral bloodleukocytes, bone marrow stem cells and embryonic stem cells. Theexpression vectors require that the pertinent sequence, i.e., thosenucleic acids described supra, be operably linked to a promoter.

As used herein, a “vector” may be any of a number of nucleic acidmolecules into which a desired sequence may be inserted by restrictionand ligation for transport between different genetic environments or forexpression in a host cell. Vectors are typically composed of DNAalthough RNA vectors are also available. Vectors include, but are notlimited to, plasmids, phagemids and virus genomes. A cloning vector isone which is able to replicate in a host cell, and which is furthercharacterized by one or more endonuclease restriction sites at which thevector may be cut in a determinable fashion and into which a desired DNAsequence may be ligated such that the new recombinant vector retains itsability to replicate in the host cell. In the case of plasmids,replication of the desired sequence may occur many times as the plasmidincreases in copy number within the host bacterium or just a single timeper host before the host reproduces by mitosis. In the case of phage,replication may occur actively during a lytic phase or passively duringa lysogenic phase. An expression vector is one into which a desired DNAsequence may be inserted by restriction and ligation such that it isoperably joined to regulatory sequences and may be expressed as an RNAtranscript. Vectors may further contain one or more marker sequencessuitable for use in the identification of cells which have or have notbeen transformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques (e.g., green fluorescent protein). Preferredvectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which theyare operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

It will also be recognized that the invention embraces the use of theDOS cDNA sequences or Mutant DOS cDNA sequences in expression vectors,as well as to transfect host cells and cell lines, be these prokaryotic(e.g., E. coli), or eukaryotic (e.g., CHO cells, COS cells, yeastexpression systems and recombinant baculovirus expression in insectcells). Especially useful are mammalian cells such as human, mouse,hamster, pig, goat, primate, etc. They may be of a wide variety oftissue types, and include primary cells and cell lines. Specificexamples include keratinocytes, peripheral blood leukocytes, bone marrowstem cells and embryonic stem cells. The expression vectors require thatthe pertinent-sequence, i.e., those nucleic acids described supra, beoperably linked to a promoter.

According to still a further aspect of the invention, a transgenicnon-human animal comprising an expression vector of the invention isprovided, including a transgenic non-human animal which has reducedexpression of a DOS nucleic acid molecule or a Mutant DOS nucleic acidmolecule elevated expression of a DOS nucleic acid molecule or a MutantDOS nucleic acid molecule.

As used herein, “transgenic non-human animals” includes non-humananimals having one or more exogenous nucleic acid molecules incorporatedin germ line cells and/or somatic cells. Thus the transgenic animalinclude “knockout” animals having a homozygous or heterozygous genedisruption by homologous recombination, animals having episomal orchromosomally incorporated expression vectors, etc. Knockout animals canbe prepared by homologous recombination using embryonic stem cells as iswell known in the art. The recombination can be facilitated by thecre/lox system or other recombinase systems known to one of ordinaryskill in the art. In certain embodiments, the recombinase system itselfis expressed conditionally, for example, in certain tissues or celltypes, at certain embryonic or post-embryonic developmental stages,inducibly by the addition of a compound which increases or decreasesexpression, and the like. In general, the conditional expression vectorsused in such systems use a variety of promoters which confer the desiredgene expression pattern (e.g., temporal or spatial). Conditionalpromoters also can be operably linked to DOS nucleic acid molecules toincrease or decrease expression of a DOS molecule in a regulated orconditional manner. Trans-acting negative or positive regulators of DOSactivity or expression also can be operably linked to a conditionalpromoter as described above. Such trans-acting regulators includeantisense DOS nucleic acid molecules, nucleic acid molecules whichencode dominant negative DOS molecules, ribozyme molecules specific forDOS nucleic acid molecules, and the like. The transgenic non-humananimals are useful in experiments directed toward testing biochemical orphysiological effects of diagnostics or therapeutics for conditionscharacterized by increased or decreased DOS molecule expression. Otheruses will be apparent to one of ordinary skill in the art. Thus, theinvention also permits the construction of DOS gene “knock-outs” incells and in animals, providing materials for studying certain aspectsof cytoskeletal organization, cell migration, cancer, and metastasis.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding DOS protein, fragment, or variantthereof. The heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen, Carlsbad, Calif.), which contains an Epstein Barrvirus (EBV) origin of replication, facilitating the maintenance ofplasmid as a multicopy extrachromosomal element. Another expressionvector is the pEF-BOS plasmid containing the promoter of polypeptideElongation Factor 1α, which stimulates efficiently transcription invitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res.18:5322, 1990), and its use in transfection experiments is disclosed by,for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Stillanother preferred expression vector is an adenovirus, described byStratford-Perricaudet, which is defective for E1 and E3 proteins (J.Clin. Invest. 90:626-630, 1992). The use of the adenovirus as anAdeno.P1A recombinant is disclosed by Warnier et al., in intradermalinjection in mice for immunization against P1A (Int. J. Cancer,67:303-310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

According to another aspect of the invention, an isolated proteinencoded by any of the foregoing isolated nucleic acid molecules of theinvention is provided. Preferably, the isolated protein comprises aprotein selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The invention also embracesMutant DOS proteins, such as those described in the Examples.

The invention also provides isolated proteins, which include theproteins of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, and 30 and unique fragments of thereof. Such proteins are useful,for example, alone or as fusion proteins to generate antibodies, as acomponent(s) of an immunoassay or for determining the bindingspecificity of HLA molecules and/or CTL clones for DOS proteins.

As used herein, a DOS protein refers to a protein which is encoded by anucleic acid having SEQ ID NO:1 or SEQ ID NO:3, a functional fragmentthereof, or a functional equivalent thereof (e.g., a nucleic acidsequence encoding the same protein as encoded by SEQ ID NO:1 or SEQ IDNO:3), provided that the functional fragment or equivalent encodes a DOSprotein which exhibits a DOS functional activity. As used herein, a DOSfunctional activity refers to the ability of a DOS protein to modulateone or more of the following parameters: cytoskeletal organization, cellgrowth, cell proliferation, cell migration, and/or cell-cellinteractions. An exemplary DOS functional activity is a tumor suppressoractivity such as suppressing and/or reducing tumor cell growth,proliferation, and/or metastasis. Although not wishing to be bound toany particular theory or mechanism, it is believed that the DOS proteinmay affect at least some of the above-noted cell functions byinteracting with actin and, thereby, modulating cytoskeletalorganization.

In one aspect of the invention a functional protein fragment of SEQ IDNO: 2 comprising SEQ ID NO: 32 is provided.

Proteins can be isolated from biological samples including tissue orcell homogenates, and can also be expressed recombinantly in a varietyof prokaryotic and eukaryotic expression systems by constructing anexpression vector appropriate to the expression system, introducing theexpression vector into the expression system, and isolating therecombinantly expressed protein. Short polypeptides, including antigenicpeptides (such as are presented by MHC molecules on the surface of acell for immune recognition) also can be synthesized chemically usingwell-established methods of peptide synthesis.

Thus, as used herein with respect to proteins, “isolated” meansseparated from its native environment and present in sufficient quantityto permit its identification or use. Isolated, when referring to aprotein or polypeptide, means, for example: (i) selectively produced byexpression of a recombinant nucleic acid or (ii) purified as bychromatography or electrophoresis. Isolated proteins or polypeptidesmay, but need not be, substantially pure. The term “substantially pure”means that the proteins or polypeptides are essentially free of othersubstances with which they may be found in nature or in vivo systems toan extent practical and appropriate for their intended use.Substantially pure proteins may be produced by techniques well known inthe art. Because an isolated protein may be admixed with apharmaceutically acceptable carrier in a pharmaceutical preparation, theprotein may comprise only a small percentage by weight of thepreparation. The protein is nonetheless isolated in that it has beenseparated from the substances with which it may be associated in livingsystems, e.g. isolated from other proteins.

A fragment of a DOS protein, for example, generally has the features andcharacteristics of fragments including unique fragments as discussedabove in connection with nucleic acid molecules. As will be recognizedby those skilled in the art, the size of a fragment which is unique willdepend upon factors such as whether the fragment constitutes a portionof a conserved protein domain. Thus, some regions of DOS proteins willrequire longer segments to be unique while others will require onlyshort segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8,9, 10, 11, and 12 amino acids long).

Unique fragments of a protein preferably are those fragments whichretain a distinct functional capability of the protein. Functionalcapabilities which can be retained in a fragment of a protein includeinteraction with antibodies, interaction with other proteins orfragments thereof, selective binding of nucleic acid molecules, andenzymatic activity. One important activity is the ability to act as asignature for identifying the polypeptide. Another is the ability toprovoke in a human an immune response to a Mutant DOS molecule but notprovoke an immune response to a DOS molecule.

Those skilled in the art are well versed in methods for selecting uniqueamino acid sequences, typically on the basis of the ability of thefragment to selectively distinguish the sequence of interest fromnon-family members. A comparison of the sequence of the fragment tothose on known data bases typically is all that is necessary.

The invention embraces variants of the DOS proteins described herein. Asused herein, a “variant” of a DOS protein is a protein which containsone or more modifications to the primary amino acid sequence of a DOSprotein. Modifications which create a DOS protein variant can be made toa DOS protein 1) to produce, increase, reduce, or eliminate—an activityof the DOS protein or Mutant DOS protein; 2) to enhance a property ofthe DOS protein, such as protein stability in an expression system orthe stability of protein-protein binding; 3) to provide a novel activityor property to a DOS protein, such as addition of an antigenic epitopeor addition of a detectable moiety; or 4) to provide equivalent orbetter binding to an HLA molecule. Modifications to a DOS protein or toa Mutant DOS protein are typically made to the nucleic acid moleculewhich encodes the protein, and can include deletions, point mutations,truncations, amino acid substitutions and additions of amino acids ornon-amino acid moieties. Alternatively, modifications can be madedirectly to the protein, such as by cleavage, addition of a linkermolecule, addition of a detectable moiety, such as biotin, addition of afatty acid, and the like. Modifications also embrace fusion proteinscomprising all or part of the DOS amino acid sequences. One of skill inthe art will be familiar with methods for predicting the effect onprotein conformation of a change in protein sequence, and can thus“design” a variant DOS polypeptide according to known methods. Oneexample of such a method is described by Dahiyat and Mayo in Science278:82-87, 1997, whereby proteins can be designed de novo. The methodcan be applied to a known protein to vary only a portion of the proteinsequence. By applying the computational methods of Dahiyat and Mayo,specific variants of a DOS protein can be proposed and tested todetermine whether the variant retains a desired conformation.

In general, variants include DOS proteins which are modifiedspecifically to alter a feature of the protein unrelated to its desiredphysiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of aDOS protein by eliminating proteolysis by proteases in an expressionsystem (e.g., dibasic amino acid residues in yeast expression systems inwhich KEX2 protease activity is present).

Mutations of a nucleic acid molecule which encode a DOS proteinpreferably preserve the amino acid reading frame of the coding sequence,and preferably do not create regions in the nucleic acid which arelikely to hybridize to form secondary structures, such a hairpins orloops, which can be deleterious to expression of the variant protein.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe protein. Variant proteins are then expressed and tested for one ormore activities to determine which mutation provides a variant proteinwith the desired properties. Further mutations can be made to variants(or to non-variant DOS proteins) which are silent as to the amino acidsequence of the protein, but which provide preferred codons fortranslation in a particular host. The preferred codons for translationof a nucleic acid in, e.g., E. coli, are well known to those of ordinaryskill in the art. Still other mutations can be made to the noncodingsequences of a DOS gene or cDNA clone to enhance expression of theprotein. The activity of variants of DOS proteins can be tested bycloning the gene encoding the variant DOS protein into a bacterial ormammalian expression vector, introducing the vector into an appropriatehost cell, expressing the variant DOS protein, and testing for afunctional capability of the DOS protein as disclosed herein. Forexample, the variant DOS protein can be tested for reaction withautologous or allogeneic sera. Preparation of other variant proteins mayfavor testing of other activities, as will be known to one of ordinaryskill in the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in DOS proteins to provide functional variantsof the foregoing proteins, i.e, the variants S which the functionalcapabilities of the DOS proteins. As used herein, a “conservative aminoacid substitution” refers to an amino acid substitution which does notalter the relative charge or size characteristics of the protein inwhich the amino acid substitution is made. Conservative substitutions ofamino acids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

For example, upon determining that a peptide derived from a DOS proteinplays a role in tumor suppression, cytoskeletal organization, cellproliferation, and/or cell migration, one can make conservative aminoacid substitutions to the amino acid sequence of the peptide. Thesubstituted peptides can then be tested for one or more of theabove-noted functions, in vivo or in vitro. These variants can be testedfor improved stability and are useful, inter alia, in pharmaceuticalcompositions.

Functional variants of DOS proteins, i.e., variants of proteins whichretain the function of the DOS proteins, can be prepared according tomethods for altering polypeptide sequence known to one of ordinary skillin the art such as are found in references which compile such methods,e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. Exemplary functionalvariants of the DOS proteins include conservative amino acidsubstitutions of proteins encoded by SEQ ID NOs:2 or 4. Conservativeamino-acid substitutions in the amino acid sequence of DOS proteins toproduce functional variants of DOS proteins typically are made byalteration of the nucleic acid molecule encoding a DOS protein (e.g. SEQID NO:1 or SEQ ID NO:3). Such substitutions can be made by a variety ofmethods known to one of ordinary skill in the art. For example, aminoacid substitutions may be made by PCR-directed mutation, site-directedmutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad.Sci. U.S.A. 82: 488-492, 1985), or by chemical synthesis of a geneencoding a DOS protein. Where amino acid substitutions are made to asmall unique fragment of a DOS protein the substitutions can be made bydirectly synthesizing the peptide. The activity of functional variantsor fragments of DOS protein can be tested by cloning the gene encodingthe altered DOS protein into a bacterial or mammalian expression vector,introducing the vector into an appropriate host cell, expressing thealtered DOS protein, and testing for a functional capability of the DOSproteins as disclosed herein.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof the DOS protein molecules. A variety of methodologies well-known tothe skilled practitioner can be utilized to obtain isolated DOSmolecules. The polypeptide may be purified from cells which naturallyproduce the protein by chromatographic means or immunologicalrecognition. Alternatively, an expression vector may be introduced intocells to cause production of the protein. In another method, mRNAtranscripts may be microinjected or otherwise introduced into cells tocause production of the encoded protein. Translation of mRNA incell-free extracts such as the reticulocyte lysate system also may beused to produce polypeptide. Those skilled in the art also can readilyfollow known methods for isolating DOS proteins. These include, but arenot limited to, immunochromatography, HPLC, size-exclusionchromatography, ion-exchange chromatography and immune-affinitychromatography.

The isolation and identification of DOS nucleic acid molecules alsomakes it possible for the artisan to diagnose a disorder characterizedby aberrant expression of a DOS nucleic acid molecule or protein. Thesemethods involve determining the aberrant expression of one or more DOSnucleic acid molecules and/or Mutant DOS nucleic acid molecules, and/orencoded DOS proteins and/or Mutant DOS proteins. In the former twosituations, such determinations can be carried out via any standardnucleic acid determination assay, including the polymerase chainreaction, or assaying with labeled hybridization probes. In the lattertwo situations, such determinations can be carried out by screeningpatient antisera for recognition of the polypeptide or by assayingbiological samples with binding partners (e.g., antibodies) for DOSproteins or Mutant DOS proteins.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from DOS proteins. A dominant negative polypeptideis an inactive variant of a protein, which, by interacting with thecellular machinery, displaces an active protein from its interactionwith the cellular machinery or competes with the active protein, therebyreducing the effect of the active protein. Dominant negativepolypeptides are useful, or example, for preparing transgenic non-humananimals to further characterize the functions of the DOS molecules andMutant DOS molecules disclosed herein. For example, a dominant negativereceptor which binds a ligand but does not transmit a signal in responseto binding of the ligand can reduce the biological effect of expressionof the ligand. Likewise, a dominant negative catalytically-inactivekinase which interacts normally with target proteins but does notphosphorylate the target proteins can reduce phosphorylation of thetarget proteins in response to a cellular signal. Similarly, a dominantnegative transcription factor which binds to a promoter site in thecontrol region of a gene but does not increase gene transcription canreduce the effect of a normal transcription factor by occupying promoterbinding sites without increasing transcription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and using standard mutagenesis techniques tocreate one or more dominant negative variant polypeptides. For example,one of ordinary skill in the art can modify the sequence of DOS proteinsby site-specific mutagenesis, scanning mutagenesis, partial genedeletion or truncation, and the like. See, e.g., U.S. Pat. No. 5,580,723and Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. The skilled artisanthen can test the population of mutagenized proteins for diminution in aselected and/or for retention of such an activity. Other similar methodsfor creating and testing dominant negative variants of a protein will beapparent to one of ordinary skill in the art.

In yet a further aspect of the invention, binding polypeptides thatselectively bind to a DOS molecule and/or to a Mutant DOS molecule areprovided. According to this aspect, the binding polypeptides bind to anisolated nucleic acid or protein of the invention, including binding tounique fragments thereof. Preferably, the binding polypeptides bind to aDOS protein, a Mutant DOS protein, or a unique fragment thereof. Incertain particularly preferred embodiments, the binding polypeptidebinds to a Mutant DOS protein but does not bind to a DOS protein, i.e.,the binding polypeptides are selective for binding to the Mutant DOSprotein and can be used in various assays to detect the presence of theMutant DOS protein without detecting DOS protein. Such Mutant DOSprotein binding polypeptides also can be used to selectively bind to aMutant DOS molecule in a cell (in vivo or ex vivo) for imaging andtherapeutic applications in which, for example, the binding polypeptideis tagged with a detectable label and/or a toxin for targetted deliveryto the Mutant DOS molecule.

In preferred embodiments, the binding polypeptide is an antibody orantibody fragment, more preferably, an Fab or F(ab)₂ fragment of anantibody. Typically, the fragment includes a CDR3 region that isselective for the DOS protein or Mutant DOS protein. Any of the varioustypes of antibodies can be used for this purpose, including monoclonalantibodies, humanized antibodies and chimeric antibodies.

Thus, the invention provides agents which bind to DOS proteins or MutantDOS proteins encoded by DOS nucleic acid molecules or Mutant DOS nucleicacid molecules, respectively, and in certain embodiments preferably tounique fragments of the DOS proteins or Mutant DOS proteins. Suchbinding partners can be used in screening assays to detect the presenceor absence of a DOS protein or a Mutant DOS protein and in purificationprotocols to isolate such DOS proteins. Likewise, such binding partnerscan be used to selectively target drugs, toxins or other molecules tocells which express Mutant DOS proteins. In this manner, cells presentin solid or non-solid tumors which express Mutant DOS proteins can betreated with cytotoxic compounds. Such agents also can be used toinhibit the native activity of the DOS polypeptides, for example, bybinding to such polypeptides, to further characterize the functions ofthese molecules

The invention, therefore, provides antibodies or fragments of antibodieshaving the ability to selectively bind to Mutant DOS proteins, andpreferably to unique fragments thereof. Antibodies include polyclonal,monoclonal, and chimeric antibodies, prepared, e.g., according toconventional methodology.

The antibodies of the present invention thus are prepared by any of avariety of methods, including administering protein, fragments ofprotein, cells expressing the protein or fragments thereof and the liketo an animal to induce polyclonal antibodies. The production ofmonoclonal antibodies is according to techniques well known in the art.As detailed herein, such antibodies may be used for example to identifytissues expressing protein or to purify protein. Antibodies also may becoupled to specific labeling agents for imaging or to antitumor agents,including, but not limited to, methotrexate, radioiodinated compounds,toxins such as ricin, other cytostatic or cytolytic drugs, and so forth.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of nonspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies. Thus, the invention involves polypeptides ofnumerous size and type that bind specifically to mutant DOS proteins.These polypeptides may be derived also from sources other than antibodytechnology. For example, such polypeptide binding agents can be providedby degenerate peptide libraries which can be readily prepared insolution, in immobilized form or as phage display libraries.Combinatorial libraries also can be synthesized of peptides containingone or more amino acids. Libraries further can be synthesized ofpeptides and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent a completely degenerate or biased array. Onethen can select phage-bearing inserts which bind to a DOS protein or aMutant DOS protein. This process can be repeated through several cyclesof reselection of phage that bind to a DOS protein or a Mutant DOSprotein. Repeated rounds lead to enrichment of phage bearing particularsequences. DNA sequence analysis can be conducted to identify thesequences of the expressed polypeptides. The minimal linear portion ofthe sequence that binds to the DOS protein or the Mutant DOS protein canbe determined. One can repeat the procedure using a biased librarycontaining inserts containing part or all of the minimal linear portionplus one or more additional degenerate residues upstream or downstreamthereof. Thus, the DOS proteins of the invention can be used to screenpeptide libraries, including phage display libraries, to identify andselect peptide binding partners of the DOS proteins of the invention.Such molecules can be used, as described, for screening assays, fordiagnostic assays, for purification protocols or for targeting drugs,toxins and/or labeling agents (e.g. radioisotopes, fluorescentmolecules, etc.) to cells which express mutant DOS genes such as cancercells which have aberrant DOS expression. Such binding agent moleculescan also be prepared to bind complexes of an DOS protein and an HLAmolecule by selecting the binding agent using such complexes.

As detailed herein, the foregoing antibodies and other binding moleculesmay be used for example to identify tissues expressing mutant protein orto purify mutant protein. Antibodies also may be coupled to specificdiagnostic labeling agents for imaging of cells and tissues withaberrant DOS expression or to therapeutically useful agents according tostandard coupling procedures. Diagnostic agents include, but are notlimited to, barium sulfate, iocetamic acid, iopanoic acid, ipodatecalcium, diatrizoate sodium, diatrizoate meglumine, metrizamide,tyropanoate sodium and radiodiagnostics including positron emitters suchas fluorine-18 and carbon-11, gamma emitters such as iodine-123,technitium-99m, iodine-131 and indium-111, nuclides for nuclear magneticresonance such as fluorine and gadolinium. Other diagnostic agentsuseful in the invention will be apparent to one of ordinary skill in theart. As used herein, “therapeutically useful agents” include anytherapeutic molecule which desirably is targeted selectively to a cellor tissue selectively with an aberrant DOS expression, includingantineoplastic agents, radioiodinated compounds, toxins, othercytostatic or cytolytic drugs, and so forth. Antineoplastic therapeuticsare well known and include: aminoglutethimide, azathioprine, bleomycinsulfate, busulfan, carmustine, chlorambucil, cisplatin,cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin,daunorubicin, doxorubicin, taxol, etoposide, fluorouracil, interferon,lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCl,thioguanine, vinblastine sulfate and vincristine sulfate. Additionalantineoplastic agents include those disclosed in Chapter 52,Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and theintroduction thereto, pp. 1202-1263, of Goodman and Gilman's, ThePharmacological Basis of Therapeutics, Eighth Edition, 1990,McGraw-Hill, Inc. (Health Professions Division). Toxins can be proteinssuch as, for example, pokeweed anti-viral protein, cholera toxin,pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, orPseudomonas exotoxin. Toxin moieties can also be high energy-emittingradionuclides such as cobalt-60.

According to a further aspect of the invention, pharmaceuticalcompositions containing the nucleic acid molecules, proteins, andbinding polypeptides of the invention are provided. The pharmaceuticalcompositions contain any of the foregoing therapeutic agents in apharmaceutically acceptable carrier. Thus, in a related aspect, theinvention provides a method for forming a medicament that involvesplacing a therapeutically effective amount of the therapeutic agent inthe pharmaceutically acceptable carrier to form one or more doses.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredients. The term “physiologically acceptable” refers toa non-toxic material that is compatible with a biological system such asa cell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712). Those of skill in the art can readilydetermine the various parameters and conditions for producing antibodyaerosols without resort to undue experimentation. When using antisensepreparations of the invention, slow intravenous administration ispreferred.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, stimulates the desired response.In the case of treating cancer, the desired response is inhibiting theprogression of the cancer. This may involve only slowing the progressionof the disease temporarily, although more preferably, it involveshalting the progression of the disease permanently. In the case ofstimulating an immune response, the desired response is an increase inantibodies or T lymphocytes which are specific for the immunogen(s)employed. These responses can be monitored by routine methods or can bemonitored according to diagnostic methods of the invention discussedherein.

Where it is desired to stimulate an immune response using a therapeuticcomposition of the invention (e.g. a Mutant DOS protein fragment whichis a unique fragment of the Mutant DOS molecule), this may involve thestimulation of a humoral antibody response resulting in an increase inantibody titer in serum, a clonal expansion of cytotoxic lymphocytes, orsome other desirable immunologic response. It is believed that doses ofimmunogens ranging from one nanogram/kilogram to 100milligrams/kilogram, depending upon the mode of administration, would beeffective. The preferred range is believed to be between 500 nanogramsand 500 micrograms per kilogram. The absolute amount will depend upon avariety of factors, including the material selected for administration,whether the administration is in single or multiple doses, andindividual patient parameters including age, physical condition, size,weight, and the stage of the disease. These factors are well known tothose of ordinary skill in the art and can be addressed with no morethan routine experimentation.

According to another aspect of the invention, various diagnostic methodsare provided. In general, the methods are for diagnosing “a disordercharacterized by aberrant expression of a DOS molecule”. As used herein,“aberrant expression” refers to either or both of a decreased expression(including no expression) of a DOS molecule (nucleic acid or protein) oran increased expression of a “Mutant DOS molecule”. A Mutant DOSmolecule refers to a DOS nucleic acid molecule which includes a mutation(deletion, addition, or substitution) or to a DOS protein molecule(e.g., gene product of Mutant DOS nucleic acid molecule) which includesa mutation, provided that the mutation results in a Mutant DOS proteinthat does not have a DOS protein functional activity. The diagnosticmethods of the invention can be used to detect the presence of adisorder associated with aberrant expression of a DOS molecule, as wellas to assess the progression and/or regression of the disorder such asin response to treatment (e.g., chemotherapy, radiation).

According to this aspect of the invention, the method for diagnosing adisorder characterized by aberrant expression of a DOS moleculeinvolves: detecting in a first biological sample obtained from asubject, expression of a DOS molecule or a Mutant DOS molecule; whereindecreased expression of a DOS molecule or the increased expression of aMutant DOS molecule compared to a control sample indicates that thesubject has a disorder characterized by aberrant expression of a DOSmolecule.

As used herein, a “disorder characterized by aberrant expression of aDOS molecule” refers to a disorder in which there is a detectabledifference in the expression levels of DOS molecule(s) and/or Mutant DOSmolecule(s) in selected cells of a subject compared to the controllevels of these molecules. Thus, a disorder characterized by aberrantexpression of a DOS molecule embraces underexpression (including noexpression) of a DOS nucleic acid molecule or a DOS protein compared tocontrol levels of these molecules, as well as overexpression of a MutantDOS nucleic acid molecule or Mutant DOS protein compared to controllevels of these molecules. Such differences in expression levels can bedetermined in accordance with the diagnostic methods of the invention asdisclosed herein. Exemplary disorders that are characterized by aberrantexpression of a DOS molecule include: various cancers and disordersassociated with abnormal cytoskeleton organization, cell proliferation,cell migration, cellular growth and development, and/or cell-cellinteraction.

In certain embodiments, the methods of the invention are to diagnose acancer including, but not limited to, biliary tract cancer, brain cancer(including glioblastomas and medulloblastomas), breast cancer; cervicalcancer; choriocarcinoma, colon cancer, endometrial cancer, esophagealcancer, gastric cancer, hematological neoplasms, including acutelymphocytic and myelogenous leukemia, multiple myeloma, AIDS associatedleukemias and adult T-cell lymphoma/leukemia, intraepithelial neoplasms,including Bowen's disease and Paget's disease, liver cancer, lungcancer, lymphomas, including Hodgkin's disease and lymphocyticlymphomas, neuroblastomas, oral cancer, including squamous cellcarcinoma, ovarian cancer, including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells, pancreaticcancer, prostate cancer, rectal cancer, renal cancer includingadenocarcinoma and Wilms tumor, sarcomas, including leiomyosarcoma,rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma, skincancer, including melanoma, Kaposi's sarcoma, basocellular cancer andsquamous cell cancer, testicular cancer, including germinal tumors(seminomas, and non-seminomas such as teratomas and choriocarcinomas),stromal tumors and germ cell tumors, and thyroid cancer, includingthyroid adenocarcinoma and medullary carcinoma. In the preferredembodiments, the methods of the invention are useful for diagnosingWilms tumor, ovarian carcinoma, renal cell carcinoma, osteosarcomafibrosarcoma, prostate cancer, colon cancer and brain cancer.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the progression of a disorder. According to theseembodiments, the methods further involve: detecting in a secondbiological sample obtained from the subject, expression of a DOSmolecule or a Mutant DOS molecule, and comparing the expression of theDOS molecule or the Mutant DOS molecule in the first biological sampleand the second biological sample. In these embodiments, a decrease inthe expression of the DOS molecule in the second biological samplecompared to the first biological sample or an increase in the expressionof the Mutant DOS molecule in the second biological sample compared tothe first biological sample indicates progression of the disorder.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the regression of a disorder. According to these embodiments,the methods further involve: detecting in a second biological sampleobtained from the subject, expression of a DOS molecule or a Mutant DOSmolecule, and comparing the expression of the DOS molecule or the MutantDOS molecule in the first biological sample and the second biologicalsample. In these embodiments, an increase in the expression of the DOSmolecule in the second biological sample compared to the firstbiological sample or a decrease in the expression of the Mutant DOSmolecule in the second biological sample compared to the firstbiological sample indicates regression of the disorder.

In certain embodiments, the diagnostic methods of the invention detect aDOS molecule that is a DOS nucleic acid molecule or a Mutant DOS nucleicacid molecule as described above. In yet other embodiments, the methodsinvolve detecting a DOS protein or Mutant DOS protein as describedabove.

Various detection methods can be used to practice the diagnostic methodsof the invention. For example, when the methods can involve contactingthe biological sample with an agent that selectively binds to the DOSmolecule or to the Mutant DOS molecule to detect these molecules. Incertain embodiments, the DOS molecule is a nucleic acid and the methodinvolves using an agent that selectively binds to the DOS molecule or tothe Mutant DOS molecule, e.g., a nucleic acid that hybridizes understringent conditions to a nucleic acid sequence selected from the groupconsisiting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, and 29. In yet other embodiments, the DOS molecule is a proteinand the method involves using an agent that selectively binds to the DOSmolecule or to the Mutant DOS molecule, e.g., a binding polypeptide,such as an antibody, that selectively binds to a protein sequenceselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, and 30.

According to still another aspect of the invention, kits for performingthe diagnostic methods of the invention are provided. The kits includenucleic acid-based kits or protein-based kits. According to the formerembodiment, the kits include: one or more nucleic acid molecules thathybridize to a DOS nucleic acid molecule or to a Mutant DOS nucleic acidmolecule under stringent conditions; one or more control agents; andinstructions for the use of the nucleic acid molecules, and agents inthe diagnosis of a disorder associated with aberrant expression of a DOSmolecule. Nucleic acid-based kits optionally further include a firstprimer and a second primer, wherein the first primer and the secondprimer are constructed and arranged to selectively amplify at least aportion of an isolated DOS nucleic acid molecule selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, and 29. Alternatively, protein based-kits are provided. Such kitsinclude: one or more binding polypeptides that selectively bind to a DOSprotein or a Mutant DOS protein; one or more control agents; andinstructions for the use of the binding polypeptides, and agents in thediagnosis of a disorder associated with aberrant expression of a DOSmolecule. In the preferred embodiments, the binding polypeptides areantibodies or antigen-binding fragments thereof, such as those describedabove. In these and other embodiments, certain of the bindingpolypeptides bind to the Mutant DOS protein but do not bind to the DOSprotein to further distinguish the expression of these proteins in abiological sample.

As used herein, a subject is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat or rodent. In all embodiments human and mouseDOS molecules and human subjects are preferred.

The biological sample can be located in vivo or in vitro. For example,the biological sample can be a tissue in vivo and the agent specific forthe tumor associated nucleic acid molecule or polypeptide can be used todetect the presence of such molecules in the hematopoietic tissue (e.g.,for imaging portions of the tissue that express the tumor associatedgene products). Alternatively, the biological sample can be located invitro (e.g., a blood sample, tumor biopsy, tissue extract). In aparticularly preferred embodiment, the biological sample can be acell-containing sample, more preferably a sample containing tumor cells.Samples of tissue and/or cells for use in the various methods describedherein can be obtained through standard methods. Samples can be surgicalsamples of any type of tissue or body fluid. Samples can be useddirectly or processed to facilitate analysis (e.g., paraffin embedding).Exemplary samples include a cell, a cell scraping, a cell extract, ablood sample, a tissue biopsy, including punch biopsy, a tumor biopsy, abodily fluid, a tissue, or a tissue extract or other methods.

The invention also provides treatment methods. As used herein,“treatment” includes preventing, delaying, abating or arresting theclinical symptoms of a disorder characterized by aberrant expression ofa DOS molecule. Treatment also includes reducing or preventing tumorcell growth, proliferation, and/or metastasis.

In general, the treatment methods involve administering an agent toincrease expression of a DOS molecule and/or reduce expression of aMutant DOS molecule. Thus, these methods include gene therapyapplications. In certain embodiments, the method for treating a subjectwith a disorder characterized by aberrant expression of a DOS molecule,involves administering to the subject an effective amount of a DOSnucleic acid molecule to treat the disorder. In yet other embodiments,the method for treatment involves administering to the subject aneffective amount of an anti-sense molecule to inhibit (reduce/eliminate)expression of a Mutant DOS nucleic acid molecule and, thereby, treat thedisorder. An exemplary molecule for inhibiting expression of a MutantDOS nucleic acid molecule is an anti-sense molecule that is selectivefor the mutant nucleic acid and that does not inhibit expression of theDOS nucleic acid molecule. Alternatively, the method for treating asubject with a disorder characterized by aberrant expression of a DOSmolecule involves administering to the subject an effective amount of aDOS protein to treat the disorder. In yet another embodiment, thetreatment method involves administering to the subject an effectiveamount of a binding polypeptide to inhibit a Mutant DOS protein and,thereby, treat the disorder. In certain preferred embodiments, thebinding polypeptide is an antibody or an antigen-binding fragmentthereof; more preferably, the antibodies or antigen-binding fragmentsare labeled with one or more cytotoxic agents

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivo genetherapy using vectors such as adenovirus, retroviruses, herpes virus,and targeted liposomes is also contemplated according to the invention.

In preferred embodiments, a virus vector for delivering a nucleic acidmolecule encoding a DOS protein is selected from the group consisting ofadenoviruses, adeno-associated viruses, poxviruses including vacciniaviruses and attenuated poxviruses, Semliki Forest virus, Venezuelanequine encephalitis virus, retroviruses, Sindbis virus, and Tyvirus-like particle. Examples of viruses and virus-like particles whichhave been used to deliver exogenous nucleic acids include:replication-defective adenoviruses (e.g., Xiang et al., Virology219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997;Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovirus(Townsend et al., J. Virol. 71:3365-3374, 1997), a nonreplicatingretrovirus (Irwin et al., J. Virol. 68:5036-5044, 1994), a replicationdefective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA92:3009-3013, 1995), canarypox virus and highly attenuated vacciniavirus derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353,1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol.Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis etal., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al.,Virology 212:587-594, 1995), and Ty virus-like particle (Allsopp et al.,Eur. J. Immunol 26:1951-1959, 1996). In preferred embodiments, the virusvector is an adenovirus.

Another preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus iscapable of infecting a wide range of cell types and species and can beengineered to be replication-deficient. It further has advantages, suchas heat and lipid solvent stability, high transduction frequencies incells of diverse lineages, including hematopoietic cells, and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. The adeno-associated virus can integrate into humancellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression. In addition, wild-type adeno-associated virus infectionshave been followed in tissue culture for greater than 100 passages inthe absence of selective pressure, implying that the adeno-associatedvirus genomic integration is a relatively stable event. Theadeno-associated virus can also function in an extrachromosomal fashion.

In general, other preferred viral vectors are based on non-cytopathiceukaryotic viruses in which non-essential genes have been replaced withthe gene of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Adenoviruses and retroviruses have been approved for human gene therapytrials. In general, the retroviruses are replication-deficient (i.e.,capable of directing synthesis of the desired proteins, but incapable ofmanufacturing an infectious particle). Such genetically alteredretroviral expression vectors have general utility for thehigh-efficiency transduction of genes in vivo. Standard protocols forproducing replication-deficient retroviruses (including the steps ofincorporation of exogenous genetic material into a plasmid, transfectionof a packaging cell lined with plasmid, production of recombinantretroviruses by the packaging cell line, collection of viral particlesfrom tissue culture media, and infection of the target cells with viralparticles) are provided in Kriegler, M., Gene Transfer and Expression, ALaboratory Manual, W. H. Freeman Co., New York (1990) and Murry, E. J.Ed. Methods in Molecular Biology, vol. 7, Humana Press, Inc., Cliffton,N.J. (1991).

Preferably the foregoing nucleic acid delivery vectors: (1) containexogenous genetic material that can be transcribed and translated in amammalian cell and that can suppress tumor cell growth and/orproliferation, and/or abnormal cytoskeletal organization, and/orabnormal cell growth, cell proliferation, cell migration, and/orcell-cell interaction in a host, and preferably (2) contain on a surfacea ligand that selectively binds to a receptor on the surface of a targetcell, such as a mammalian cell, and thereby gains entry to the targetcell.

Various techniques may be employed for introducing nucleic acidmolecules of the invention into cells, depending on whether the nucleicacid molecules are introduced in vitro or in vivo in a host. Suchtechniques include transfection of nucleic acid molecule-CaPO₄precipitates, transfection of nucleic acid molecules associated withDEAE, transfection or infection with the foregoing viruses including thenucleic acid molecule of interest, liposome mediated transfection, andthe like. For certain uses, it is preferred to target the nucleic acidmolecule to particular cells. In such instances, a vehicle used fordelivering a nucleic acid molecule of the invention into a cell (e.g., aretrovirus, or other virus; a liposome) can have a targeting moleculeattached thereto. For example, a molecule such as an antibody specificfor a surface membrane protein on the target cell or a ligand for areceptor on the target cell can be bound to or incorporated within thenucleic acid molecule delivery vehicle. Especially preferred aremonoclonal antibodies. Where liposomes are employed to deliver thenucleic acid molecules of the invention, proteins which bind to asurface membrane protein associated with endocytosis may be incorporatedinto the liposome formulation for targeting and/or to facilitate uptake.Such proteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acid molecules into cells, as is known by those skilled in theart. Such systems even permit oral delivery of nucleic acid molecules.

The invention provides various research methods and compositions. Thus,according to one aspect of the invention, a method for producing a DOSprotein is provided. The method involves providing a DOS nucleic acidmolecule operably linked to a promoter, wherein the DOS nucleic acidmolecule encodes the DOS protein or a fragment thereof; expressing theDOS nucleic acid molecule in an expression system; and isolating the DOSprotein or a fragment thereof from the expression system. Preferably,the DOS nucleic acid molecule has SEQ ID NO:1 or SEQ ID NO:3. Accordingto yet another aspect of the invention, a method for producing a MutantDOS protein is provided. This method involves: providing a Mutant DOSnucleic acid molecule operably linked to a promoter, wherein the MutantDOS nucleic acid molecule encodes the Mutant DOS protein or a fragmentthereof; expressing the Mutant DOS nucleic acid molecule in anexpression system; and isolating the Mutant DOS protein or a fragmentthereof from the expression system. Preferably, the Mutant DOS nucleicacid molecule has SEQ ID NO:1 or SEQ ID NO:3 with one or more deletions,additions, or substitutions to encode a Mutant DOS protein.

The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents which mimic thefunctional activity of a DOS molecule. Such DOS functional activitiesinclude tumor suppression, cytoskeletal organization, and cellmigration. Generally, the screening methods involve assaying forcompounds which modulate (up- or down-regulate) a DOS functionalactivity.

A wide variety of assays for pharmacological agents can be used inaccordance with this aspect of the invention, including, labeled invitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays, cell-based assays such as two- or three-hybridscreens, expression assays, etc. The assay mixture comprises a candidatepharmacological agent. Typically, a plurality of assay mixtures are runin parallel with different agent concentrations to obtain a differentresponse to the various concentrations.

Typically, one of these concentrations serves as a negative control,i.e., at zero concentration of agent or at a concentration of agentbelow the limits of assay detection. Candidate agents encompass numerouschemical classes, although typically they are organic compounds.Preferably, the candidate pharmacological agents are small organiccompounds, i.e., those having a molecular weight of more than 50 yetless than about 2500, preferably less than about 1000 and, morepreferably, less than about 500. Candidate agents comprise functionalchemical groups necessary for structural interactions with proteinsand/or nucleic acid molecules, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups and more preferably at least three of thefunctional chemical groups. The candidate agents can comprise cycliccarbon or heterocyclic structure and/or aromatic or polyaromaticstructures substituted with one or more of the above-identifiedfunctional groups. Candidate agents also can be biomolecules such aspeptides, saccharides, fatty acids, sterols, isoprenoids, purines,pyrimidines, derivatives or structural analogs of the above, orcombinations thereof and the like. Where the agent is a nucleic acidmolecule, the agent typically is a DNA or RNA molecule, althoughmodified nucleic acid molecules as defined herein are also contemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease, inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

An exemplary binding assay is described herein. In general the mixtureof the foregoing assay materials is incubated under conditions whereby,but for the presence of the candidate pharmacological agent, the DOSmolecule or the Mutant DOS molecule specifically binds the binding agent(e.g., antibody, complementary nucleic acid). The order of addition ofcomponents, incubation temperature, time of incubation, and otherparameters of the assay may be readily determined. Such experimentationmerely involves optimization of the assay parameters, not thefundamental composition of the assay. Incubation temperatures typicallyare between 4° C. and 40° C. Incubation times preferably are minimizedto facilitate rapid, high throughput screening, and typically arebetween 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe DOS molecule or the Mutant DOS molecule and one or more bindingagents is detected by any convenient method available to the user. Forcell free binding type assays, a separation step is often used toseparate bound from unbound components. The separation step may beaccomplished in a variety of ways. Conveniently, at least one of thecomponents is immobilized on a solid substrate, from which the unboundcomponents may be easily separated. The solid substrate can be made of awide variety of materials and in a wide variety of shapes, e.g.,microtiter plate, microbead, dipstick, resin particle, etc. Thesubstrate preferably is chosen to maximum signal to noise ratios,primarily to minimize background binding, as well as for ease ofseparation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromotograpic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. For cell free binding assays, oneof the components usually comprises, or is coupled to, a detectablelabel. A wide variety of labels can be used, such as those that providedirect detection (e.g., radioactivity, luminescence, optical or electrondensity, etc). or indirect detection (e.g., epitope tag such as the FLAGepitope, enzyme tag such as horseseradish peroxidase, etc.). The labelmay be bound to a DOS binding partner (e.g., polypeptide), orincorporated into the structure of the binding partner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,strepavidin-biotin conjugates, etc. Methods for detecting the labels arewell known in the art.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1

A. Introduction

The accumulation of somatic mutations in tumor cells provides selectiveadvantage which results in cancer progression and metastasis.Characterization of these genetic events in cancer progression isessential for the understanding of tumor biology and identification ofnovel therapeutic targets. Representational difference analysis (RDA) isa powerful technique which has been successfully used to identify tumorsuppressor genes by mapping homozygous deletions. However, RDA on humantumors is limited by common polymorphisms. Gene deletions occur ininbred mouse tumor models, which can be readily isolated by RDA becausethese strains have low prevalence of polymorphisms. Human orthologs ofmouse tumor suppressor genes are then identified using gene predictionprograms.

On the basis of this hypothesis a homozygous deletion was identified onmouse chromosome 12 in an osteosarcoma cell line derived from a NF2/p53heterozygous mouse. This region is syntenic to a sequenced contig onhuman chromosome 7q31. With the assistance of gene prediction programs,DOS (Deleted in Osteosarcoma), a novel gene encoding a protein of 1966amino acids was cloned. The protein has sequence homology to three knownproteins in the database, namely DOCK180, myoblast city and Ced 5. Thesegenes have been implicated in the regulation of actin cytoskeletonremodeling and integrin signaling. Since DOS appears to be deleted inmouse osteosarcoma, we believe DOS is a tumor suppressor gene and/or isinvolved in regulation of actin cytoskeleton reorganization and cellmigration (e.g., as a negative regulator).

Sceening for Mutations in DOS in Mouse Cancer

We have available to us fifty cancer cell lines derived from p53 andNF2/p53 mouse tumor model to validate the involvement of DOS mutationsin mouse tumorigenesis. These cell lines are screened for mutations bysequencing. We believe that DOS is frequently targeted by mutations inthe p53 and NF2/p53 model systems and/or is mutated in other mouse tumormodels, which can be confirmed by including tumors from other geneticbackgrounds.

Screening for Mutations in DOS in Human Cancer

The DOS cDNA in twenty-five human cancer cell lines derived from a widerange of tumors is sequenced to determine if DOS is mutated in humancancer. The DOS in sporadic human cancer and matched normal specimens isalso sequenced to search for loss of function mutations. Furthermore,whether DOS mutations occur early or late in carcinogenesis isinvestigated by corroborating mutations with histopathology.

Biochemical and Functional Characterization of DOS

DOS belongs to a family of proteins that affect morphogenesis and cellmigration. To further characterize the role of DOS in such fundamentalprocesses, epitope tagged, full length and C-terminus DOS mutantbacterial and mammalian expression constructs are prepared forfunctional studies. In particular, the osteosarcoma cell line with adeletion in DOS is useful in functional studies since it lacks thenative protein. Antibodies to DOS are produced to performimmunoprecipitation and immunoblotting studies. Signal transductionstudies focussing on the reorganization of actin cytoskeleton via Rho,Rac and Cdc42 are performed. Protein interaction studies are performedby GST pull down assays with SH3 domains of proteins such as Crk, CAS,Src and Nck. The role of DOS in signaling (e.g., integrin signaling),cell migration, proliferation, transformation and apoptosis is furthercharacterized.

Strategies to Detect Allelic Loss in Tumors

Significant technological advances have been made to identify regions ofchromosomes involved in tumor progression. Analyses of metaphasechromosomes show chromosomal rearrangements in leukemia and lymphomas(10). This is more difficult in solid tumors where karyotyping is lesscommonly performed. Fluorescence in situ hybridization (FISH) hasgreatly improved the sensitivity and specificity of detecting chromosomeaberrations (11, 12). However, its application in human malignancies isstill limited because of complex karyotypes seen in clinical samples.Comparative genome hybridization (CGH) uses both normal and tumorgenomes to identify regions in tumor DNA that have undergone changes incopy number (13). In this technique, normal and tumor DNA are labeledwith two different haptens that fluoresce at different wavelengths. Theprobes are then hybridized to metaphase chromosomes in the presence ofexcess Cot-1 DNA thus inhibiting hybridization of labeled repetitivesequences. The ratio of the amount of two genomes that hybridize tospecific areas of the chromosomes indicates the copy number of the twosamples. CGH is currently limited to a resolution of 10 to 20 Mb andmore sensitive in detecting amplifications rather than a small deletion(9).

RDA as a Technology to Study Cancer Genetics

RDA, a PCR based subtractive hybridization technique, is particularlyapplicable in isolating homozygous deletions in tumors (14, 18, 19). Ithas already been successful in isolating tumor suppressor genes PTEN andDMBT1 and has played a significant role in cloning of BRCA2 (15, 16,17). A detailed description of RDA follows.

In principle, a homozygous deletion in tumor DNA (also referred astarget sequence in this proposal) should be readily identified bysubtractive hybridization with normal DNA. Unfortunately, traditionalsubtractive hybridization techniques have had limited success becausethe mammalian genome is complex, not allowing sufficient time for singlecopy sequences to anneal with their respective “partners”. Thus, only 10to 100 fold enrichment of target sequence can be obtained which haslimited its use in tumor genetics.

Lisistyn et al. (14) described a technique called RepresentationalDifference Analysis (RDA) in which 10⁵ to 10⁶ fold enrichment of targetsequences can be obtained after two or three rounds of hybridization.This makes it possible to isolate minor differences such as a smalldeletion in complex mammalian genomes. The first step in RDA is toisolate DNA from two closely related genomes such as a DNA from normaland tumor cells from the same patient. Normal DNA (referred to as“tester”) and the tumor DNA (referred to as “driver”) are digestedseparately with a restriction enzyme such as Bgl II. This creates anassortment of different sized fragments. Adaptors with cohesive Bgl IIends are then ligated to the ends of the Bgl II digested fragments.These adaptors called “R” adaptors are comprised of 24mer and 12meroligonucleotides annealed together creating a Bgl II cohesive end. Afterthe ligation, the ends of the fragments are filled in with Taqpolymerase, and then undergo 20 cycles of PCR using the 24meroligonucleotide as the PCR primer. This 20 cycle PCR product is calledthe amplicon. Because PCR preferentially amplifies smaller fragments,only fragments of one kilobase or less will be amplified. Consequently,the complexity of the genome is reduced and only a “representation” ofthe genome remains, specifically—Bgl II fragments smaller than 1 kb.

For both the tester and driver amplicons, each are separately digestedwith Bgl II to remove the R-adaptors. For the tester amplicon alone, anew set of adaptors, the J-adaptors are ligated to the Bgl II cohesiveends of the tester amplicon. They are then annealed with the driveramplicon (lacking adaptors) in a 1:100 ratio. Three differentpopulations of annealed duplex DNA form; 1) driver:driver 2)driver:tester 3) tester:tester. The ends of the annealed DNA are thenfilled in and PCR is performed for 10 cycles using the J-24mer as thePCR primer. In this PCR step only the tester:tester DNA duplex will beamplified exponentially. The driver:driver duplex lacks the ligated endsand will not be amplified, and the driver:tester duplex will onlyundergo linear amplification since only one end of the duplex containsthe ligated 24mer. The PCR products then undergo digestion with mungbean nuclease to remove any single stranded DNA, and then an additional20-25 cycles of PCR are performed. This subtraction/hybridizationamplification is repeated two more times to further eliminate amplifiednon-specific background products. In the second and third round theprocess of “kinetic enrichment”, i.e. increase in the concentration oftarget sequences relative to non-specific background sequences that havebeen amplified, further enhances the enrichment of target sequences. Asthe relative concentration of the target sequences increases, thekinetics of annealing at the hybridization step also increases.Consequently, enrichment in the second and third round have twocomponents-subtraction, and increased kinetic annealing rates. RDAenriches for target sequences an estimated 10⁶ fold (18).

Tumor Specimens for RDA

The selection of appropriate starting material is believed to be animportant step in RDA. Extremely pure DNA samples from normal and cancercell are needed since contaminating normal cells within a tumor maybecome significant when tumor DNA is in excess compared with tester DNA.Using DNA from tumor and normal tissue from the same patient, webelieve, is essential to reduce polymorphic differences. If sufficientamount of genomic DNA is available, Southern bolt analysis is performedto distinguish homozygous deletions from RFLP with LOH. Thus, humantumor cells in culture are excellent specimens for RDA (19). Once atumor suppressor gene is identified, these cells are used to carry outfunctional studies. Unfortunately, for most cancer cell lines, normalcells from the same patient may not be available. In such instances,freshly isolated tumor samples is an excellent source for both testerand driver DNA because resections include normal tissue surrounding thetumor. However, the tumor bed usually contains normal stroma withendothelial and inflammatory cells. Transplanting these tumors in micei.e. heterotransplants or xenografts, for >2 passages provides arelatively pure population of cancer cells and circumvents this problem.Mouse stromal DNA contamination of the driver is not a concern in RDA.Alternatively, laser capture micro-dissection can be used to isolate apure population of cells directly from tumor (22). The use of OTC forfreezing specimens allows efficient PCR amplification aftermicro-dissection, which has been reported to be a problem in paraffinembedded specimens. However, the downstream analysis of subtractedclones (e.g. Southern blots) may become difficult because of a limitedamount of DNA and the absence of appropriate cell lines for functionalstudies.

Mouse models of human cancer are a powerful tool for RDA analysis, andhave not been explored to date. A number of knock out models mimic humantumors and genetic lesions such as chromosomal aberrations and LOH havebeen reported during tumor progression (24, 25, 26). Tumor cells can becultured easily and we use tail DNA from the same mouse as tester. Ofgreat importance is the absence of polymorphisms within inbred mousestrains. We believe that this feature dramatically enhances the abilityto detect pathological deletions in cancer.

B. Results

RDA Analysis

Salient features in RDA are the preparation of the amplicon DNAs andhybridization of normal amplicon with tumor amplicon in a molar ratio of1:100 followed by PCR amplification of re-annealed normal DNA sequences.Amplicons were prepared with genomic DNA from both cells in culture andxenogrrafts as described by Lisitsyn and Wigler, 1995 (19). Samplesobtained from laser capture micro-dissection were prepared as describedby Michiels et. al., 1998 (23).

The general scheme for evaluating the PCR fragments obtained from the3rd round of RDA is as follows. Third round RDA products are cloned andinserts are used to probe a Southern blot containing both tester anddriver amplicon DNA. Clones that hybridize to both normal and tumoramplicons represent unsubtracted background. This is variable dependingon individual subtractions and background clones can be as high as50-75% of all third round PCR products. True subtracted products i.e., aclone that hybridizes selectively to the normal DNA amplicon, can be aresult of either a homozygous deletion (a complete loss from tumorgenome) or more frequently a result of LOH i.e., presence of apolymorphic Bgl II restriction site in one allele combined with allelicloss (LOH) in the tumor of the Bgl II site present in the second allele.These LOH products are distinguished from homozygous deletions onSouthern blots using genomic DNA. Alternatively, the subtracted clone issequenced and used to design PCR primers allowing amplification ofgenomic DNA from tumor cells in LOH cases but not in cases withhomozygous deletions. This method is rapid, reliable and requires only aminute amount of genomic DNA. Therefore, it is suitable for lasercapture micro-dissected specimens. RDA was successfully performed onseveral human tumors samples derived from cells in culture, tumorxenografts grown in nude mice and laser capture micro-dissectedspecimens. Wilms tumor, ovarian carcinoma and renal cell carcinomaresults are summarized in Table 1. TABLE 1 Summary of RDA results onhuman tumors NUMBER OF TUMOR NUMBER OF HOMOZYGOUS SPECIMEN LOH DELETEDTYPE SAMPLE PRODUCTS PRODUCTS Cultured cell lines Renal Cell 5 12Carcinoma Wilms 96W 5 1 Tumor xenografts Wilms #8 6 0 Wilms #14 8 0Wilms #11 1 0 Wilms #15 2 0 Laser Capture Ovarian #1 8 1Micro-Dissection Ovarian #2 2 0 Ovarian #3 0 0 Ovarian #4 5 Notdetermined Wilms #7107 1 0

Subtracted products validated by amplicon blots were seen in 10 of 11tumor samples (ovarian carcinoma # 3 had none) implying that RDA workedin nearly all cases studied. The number of probes tested for subtractionfrom each tumor sample ranged from 4 to 20. PCR products representinghomozygous deletions were fewer than the LOH products except in the caseof renal cell carcinoma cell line. LOH products were not analyzedfurther.

Evaluation of Homozygous Deletions

Clones representing homozygous deletions were likely to contain a tumorsuppressor gene. The chromosomal location of these clones were mapped byusing one of the following: genomic database search, gene specific PCRon a radiation hybrid panel or on mouse/human somatic cell hybrids andFISH. Of the twelve probes representing homozygous deletions in theRenal Cell carcinoma cell line, five mapped to Xq22-q26. This was a verylarge deletion (>10 Mb) and was not further pursued given the largenumber of genes within this locus. The remaining 7 probes all mapped tothe locus at chromosome 9p that contain the p16^(ink4A) tumor suppressorgene, known to be frequently deleted in human cancers. Although notnovel, this result confirmed our ability to identify a homozygousdeletion in a tumor suppressor gene locus.

PCR product representing a homozygous deletion in Wilms tumor cell line96W mapped to 6q21 by FISH. Further characterization of the deletionrevealed that it represents a common polymorphism in the population(allele frequency: 0.41, homozygous deletion frequency: 0.17).Similarly, PCR products representing homozygous deletion in lasercapture micro-dissected ovarian carcinoma sample #1 was analyzed. Itmapped on chromosome 17q22, and a small, <2 Kb, homozygous deletion wasidentified. Remarkably, this region also proved to represent polymorphicdeletion in the human population (allele frequency: 0.17, homozygousdeletion frequency: 0.03.

As evident by these results, even if performed on optimal humanspecimens, frequent polymorphic deletions were present in the humanpopulation. Unlike restriction enzyme polymorphisms, these are notreadily excluded by RDA results, and require extensive analysis beforethey can be discarded (21). To circumvent the complications associatedwith human polymorphisms, we elected to work with the mouse cancermodels using syngeneic strains. Our focus on mouse tumor suppressor lociallowed us to overcome the above described complications associated withhuman polymorphisms.

RDA in Mouse Tumor Models

RDA on an inbred system, such as mouse tumor cell lines, may decreasethe probability of isolating polymorphic deletions. Mouse tumorsaccumulate sequential genetic lesions as seen in humans: mutations andallelic loss affecting both p53 and RB have been reported as havehomozygous deletions of p16 (25, 26, 27). Loss of p53 in the mouseknockout model reportedly causes genetic instability leading to avariety of tumor types (28). This represents an optimal starting pointin the selection of mouse tumor samples for genome wide analysis toisolate novel genes implicated in cancer progression.

We previously have shown that mice lacking both p53 and NF2 developlarge tumors, primarily osteosarcomas, fibrosarcomas and hepatocellularcarcinomas (29). NF2 and p53 are present on the same chromosome in themouse, and NF2/p53 double heterozygotes survive for only five monthsbefore developing multiple tumors with LOH spanning both NF2 and p53loci (29). Tumors in mice usually do not show aggressive behavior ormetastasize, whereas tumors driven by loss of both p53 and NF2 arehighly invasive and metastasize (29). We have cultured cells isolatedfrom primary and metastatic tumors and have used those cells in ourexperiments.

As a first mouse experiment, DNA was isolated from two cancer cell lines(osteosarcoma 3442 and fibrosarcoma 3085, both are derived from p53/NF2heterozygous mice) and tail DNA from SV129. A total of 18 subtractedclones were obtained from these cell lines after two rounds of RDA. All18 clones were tested by PCR for homozygous deletions. All 18 clonesrepresented restriction fragment length polymorphisms. This result showsthat like human tumors there indeed is LOH in mouse tumors.Polymorphisms arise in what is believed to be an inbred population likelaboratory mice after several generations.

To minimize population effects, tail DNA from the same mouse from whichthe tumor cell line was derived was used in RDA. The results from sevenmatched tail experiments are summarized in Table 2.

The genetic makeup of cancer cell line 3452 is as follows: the p53 andNF2 knock out alleles are present in trans on mouse chromosome 11.Products after three rounds of RDA were cloned and two of the fourclones (#1 and #4) were subtracted on amplicon blots. These clones werethen tested on a mouse Southern blot with normal tail DNA and DNA fromfour cancer cell lines digested with Bgl II. The blot was probed withboth clones #1 and #4. Since genomic DNA was digested with Bgl II forboth RDA and Southern blot, a band of the same size as the probe isexpected and is seen in normal DNA and DNA from two cancer cell lines3081 and 3678. No hybridization is seen with tumor DNA 3452. A similarresult was seen with clone #4 confirming that both clones #1 and #4 arehomozygous deletions in tumor 3452. Sequencing and blast search forthese clones did not show any matches on the sequence database.Radiation hybrid mapping was performed to determine the chromosomallocation of these clones. Clone 1 mapped to a marker, D4MIT300 on mousechromosome 4 close to the interferon locus and Cdk2na locus. The Cdk2nalocus encodes for the tumor suppressor gene p16 and deletions of thesegenes are commonly seen in several tumor types. These deletions oftentend to be large and it would not be surprising that in tumor 3452 thereis a large deletion, which inactivates p16 and the surrounding markerD4Mit300. To confirm this hypothesis, the presence of p16 exon 2 wastested in cancer cell line 3452. A p16 specific, 300 bp band is seenwhen tail DNA is used from SV 129 in PCR. Similarly, DNA from cancercell lines 3081, 3775 and 3085 amplify a p16 specific band. When tailDNA from 3452 is used p16 amplification is seen, however, p16 is notamplified when DNA from cancer cell line 3452 is used. This suggeststhat there is a somatic loss of p16 in genome of tumor 3452. Since p16is an established tumor suppressor gene, tumor cell line 3452 was notpursued any further. The results confirmed our hypothesis that mouse RDAis an optimal technique for isolating homozygous deletions leading tothe identification of tumor suppressor genes. TABLE 2 Summary of RDAresults on mouse tumors with matched tails Number of deleted Tumor cellline Sex products Result 3452 male 2 Deletion of p16 3081 female 4Deletion of a novel gene 3442 female 0 No deletions 3085 female 0 Nodeletions 3872 male 6 Y chromosome loss 3775 female 4 Not determined3678 female 0 No deletionsDeletion in Osteosarcoma Cell Line 3081

The next question to answer was that can novel tumor suppressor genes beidentified using mouse RDA approach? The results from previousexperiments show that not all NF2/p53 derived cancer cell lines lose p16and therefore could be suitable for RDA to identify novel genes. DNAfrom an osteosarcoma cell line 3081. No hybridization of the probe isseen with Bgl II digested DNA from tumor 3081. Radiation hybrid mappingshows that Clone 11 is located close to a marker D12Mit148 on mousechromosome 12. No known tumor suppressor genes are located on mousechromosome 12. The deleted clones were sequenced and used for blasthomology search of the high throughput genome sequence database wherework in progress sequences from mouse and human genome are posted. Clone11 mapped to mouse BAC clone AC 079370 and clone 13 mapped to a mouseBAC AC 079369. Both BACs mapped to mouse chromosome 12 and coveredapproximately 500,000 base pairs. The exact distance between the clones11 and 12 was not determined since the BAC sequences are currentlyunordered.

To approximate the extent of the deletion in tumor cell line 3081, themarkers surrounding D12Mit148 on chromosome 12 were examined. On the 5′end of BAC AC 069370, a Zinc finger gene ZNF277 is present. A probespecific for this gene was used in a Southern Blot on Bgl II digestedcancer cell line DNA. The results confirm that the ZNF277 gene ispresent in cancer cell line 3081. Similarly, the DLD (dihydrolipomatedehydrogenase, an E3 enzyme involved in the Kreb's cycle) gene ispresent towards the 3′ end of the deletion (3′ to BAC AC 069369). Itspresence was ascertained by PCR. A 500 bp band specific to DLD gene isamplified from all three tumors confirming the presence of DLD in tumor3081. The DLD gene is several hundred thousand base pairs from BAC069369 and the sequence data from this region is currently unavailableon public databases. We believe that there are possibly 5-6 BACs betweenthe two regions. Further blast searches revealed that BAC 084316 is just3′ to BAC 069369 and a probe specific to it was used for Southern blotanalysis. There is no hybridization of the BAC 084316 specific probe totumor DNA 3081 suggesting that it is included in the genomic deletion onchromosome 12. We have not been able to further refine the ends of thedeletion but it is clear from our data that the deletion on chromosome12 in osteosarcoma cell line 3081 involves at least three BACs spanninga region of >500,000 bp.

From Deletion to a Novel Candidate Tumor Suppressor Gene

Using blast search, we identified a DNA contig on human chromosome 7which is homologous to the deletion on mouse chromosome 12. Human 7q31is extensively involved in LOH in several tumor types. We embarked onthe positional cloning of the gene on human 7q31, focusing on theregion, which is, homologous to the deletion in mouse osteosarcoma cellline 3081.

The human 7q31 contig is sequenced and therefore can be used as inputdata in gene prediction programs. Using such programs, we were able topredict a transcript made by fusion of several exons spanning about500,000 bp genomic DNA. The start of transcription was predicted at BACAC 003077 and terminates at BAC AC 005047. The 3′ end was confirmed bysearching the EST database, which revealed several matches from multipletissue types. These ESTs are 3′ end sequence reads from libraries thatwere made by oligo-dT priming method, thus they represent 3′ ends oftranscripts. Using RT-PCR with primers from predicted exons we were ableto amplify the predicted cDNA. Since, gene prediction programs are notable to predict the 5′ ends of transcripts, we performed RACE using ahuman placenta RACE library and reverse primers from the predicted 5′end. The RACE results show that the transcription of this gene starts atBAC AC004111.1. This approach has enabled us to identify a novel gene,which is referred to as DOS for Deleted in Osteosarcoma. DOS is locatedon chromosome 7q31, with ZNF277 being telomeric while the DLD genecentromeric to it. The BAC AC 004001.1 is entirely an intron, andcontains the LOH marker D7S523. A tissue Northern blot with a DOS probeshows that a transcript of 8.5 Kb is multiple tissue types. The level ofexpression is low and can be detected by RT-PCR from several differenttissue types. The DOS mRNA encodes for a large protein of 1966 aminoacids with predicted size of 225 KD. DOS has sequence homology to threeproteins in the database, namely human DOCK180, Drosophila myoblast cityand C. elegans Ced-5. These proteins are conserved in sequence and infunction. They reportedly are involved in the initiation of cellmigration by triggering actin polymerization at the cell edge via smallGTPase Rac1. Therefore, these proteins have been reported to have a rolein several fundamental biological processes such as morphogenesis, cellmigration during development and engulfment of apoptotic cells. DOS,like DOCK180, has an N terminal SH3 domain and two conserved PFAM B(Sanger Centre, Cambridge, UK) domains namely, 9939 and 2818, suggestingextensive sequence identity. Additionally, there are regions in DOSwhere sequences are not homologous to DOCK180 such as the C terminal 300amino acids. Both proteins have proline rich regions but these regionsdo not align with each other. We believe that DOS is involved in theregulation of actin polymerization, but is distinct in function fromDOCK 180, and exhibits other functional activities that are related toits roles as a tumor suppressor gene and/or regulator of cytoskeletalorganization, cell growth, cell proliferation, cell migration, and/orcell-cell interactions.

Osteosarcoma cell line 3081 was derived from a primary tumor in anNF2/p53 heterozygous mouse. Our data show that DOS is deleted in thisparticular cell line. It is likely that DOS will be targeted bymutations in other tumor isolates arising in the same genetic backgroundsince they have similar temporal relationship to tumor progression. IfDOS is involved in cancer progression in general, it will be targeted bytumors of different genetic backgrounds.

Screening for DOS Mutations in Mouse NF2/p53 Cancer Cell Lines

About 50 cell lines derived from tumors developed in mice heterozygousfor tumor suppressor genes p53 and NF2 and both NF2 and p53 have beenisolated. Sequence analysis of DOS in these three different backgroundsis performed to determine the relative contributions of p53 and NF2loss. Mice that are heterozygous for NF2 alone form tumors at about 9months age. Mice heterozygous for both NF2 and p53 develop aggressivetumors and die by three months age. This is presumed to be due to the“genomic instability” acquired after p53 loss. Therefore, we believethere are more mutations in DOS when NF2 loss is coupled with p53 loss.

Screening for DOS Mutations Metastatic Verses Primary Cell Lines

We selected NF2/p53 tumor cell lines that have been derived from primarytumors and an equal number of cell lines derived from metastatic tumorsto confirm that DOS is involved in tumor progression. Assuming that DOSfunctions as a tumor progression gene, we anticipate observing a higherfrequency of mutations in DOS in metastatic cell lines.

Identification of the Boundaries of the Deletion in Tumor Cell Line 3081

Our results show that the 5′ end of the deletion is near the start ofthe DOS gene. Monthly homology searches are performed to find DNAsegments 3′ to the DOS gene and 5′ to the DLD gene and, thereby, furtherdefine the boundaries of this deletion. These segments are tested onmouse Southern blots. This assists in cloning the breakpoint anddetermines the contribution of such a genetic loss in tumor biology.

D. Methods to Screen for Mutations in DOS in Human Cancer

Introduction and Hypothesis

Allelic losses in tumors are typically detected as “loss ofheterozygosity” or “LOH”. While LOH is a common event in cancer, it onlyallows rough mapping of tumor suppressor loci (9). However, thesestudies do suggest that a tumor suppressor gene is nearby the LOHmarker. Human DOS is located on 7q31, a region observed to have LOH inmultiple tumor types including ovarian, prostate and breast cancer. Astudy on 22 primary ovarian cancers with 16 microsatellite markersshowed that the minimal region of LOH was at 7q31.1 involving markerD7S523. Based on mouse deletion of DOS and human LOH studies, we believethat DOS is a tumor suppressor gene and is targeted by mutations inseveral tumor types.

Sequencing of DOS cDNA in Human Cancer Cell Lines

Available to us are a panel of human cancer cell lines obtained fromeither ATCC or NCI. Multiple samples are run in a very short period oftime on Applied BioSystems 3100 Genetic analyzer (Applied Biosystems,Foster City, Calif.). Direct sequencing of RT-PCR fragments is themethod of choice as DOS is a large gene with several small exons(average size 100 bp), therefore genomic DNA sequencing is not feasible.We perform primary RT-PCR using primers spanning the entire open readingframe of DOS with cDNA from various cancer cell lines. These primary PCRreactions are diluted, then used in secondary PCR reactions. A total of13 secondary PCR reactions with an average size of 600 bp are requiredto accurately analyze every base pair of the open reading frame.Sequencing is performed on both strands using Dye-Terminator (AppliedBioSystems, Foster City, Calif.) since we are primarily interested inhomozygous changes. We anticipate two classes of mutations, one leadingto premature chain termination and the other causing missense mutations.Premature termination almost always undoubtedly causes loss of functionin gene expression and such results further validate DOS as a tumorsuppressor gene. Missense mutations are more difficult to interpretsince they represent single nucleotide polymorphism. The region ofmissense mutation in DOS from normal controls is sequenced eliminatethis possibility. DNA and RNA from EBV immortalized normal peripheralleukocytes also are used in such studies. Missense mutations are likelyto target conserved amino acid residues in mouse and human DOS. Searchfor mutations in cancer cell lines have several advantages in that cellsderived from multiple tumor types can be screened rapidly. RNA from celllines is relatively pure, free from contaminating normal tissue.

Screening for Mutations in DOS in Primary Human Tumors

A large tumor bank with most tumor types is available for our geneticstudies. For most tumors, matched normal samples are available. SinceDOS is a large gene, RNA isolated from laser capture micro dissectedmaterial is not particularly useful for RT-PCR. Surgical resections isthe method of choice. Tumors are dissected from surrounding normaltissues and RNA is isolated using STAT-60. Primary and secondary RT-PCRreactions for sequencing are performed. If a mutation is seen in aparticular sample, the matched normal is also sequenced in the sameregion. Sequencing normal genetic material from the same patient showsthat the mutation is an acquired sporadic event.

Screening for Mutations in DOS in Human Primary Verses Metastatic Tumors

Whether these mutations are acquired early or late in carcinogenesis isnext determined. For example, following identification of mutations inDOS in prostate cancer, the mutations are correlated with grade, stageand metastatic disease. Such results are consistent with a frequency ofLOH at 7q31 in prostate cancer is higher in late stage tumors andcorrelates with aggressive behavior and metastasis.

E. Method for Biochemical and Functional Characterization of DOS

Introduction and Hypothesis

Initiation of cell migration is characterized by rapid actinpolymerization to the migrating cell's leading edge. The result is aprotrusion of a lamellipodium with membrane ruffles observed at the cellsurface. These ruffles serve as sites for actin polymerization,endocytosis, protease activation and receptor tyrosine kinase signaling.Genetic and biochemical studies have shown that membrane rufflingrequires at least three proteins, namely, Rac1, DOCK180 and CrkII. Theprotein DOCK180 was originally identified by its ability to interactwith adaptor protein CrkII. It is now believed to signal downstream theintegrin receptor α_(v)β₅ and initiate cell migration. In C elegans,there are at least six genes identified in engulfment of apoptoticcells; three of these genes, Ced-2, Ced-5 and Ced-10 are a part of onecomplementation group. Ced-5 codes for the ortholog of DOCK180, Ced-2codes for ortholog of CrkII and Ced-10 encodes for Rac1. Myoblast city,a homologue of DOCK180 in Drosophila has been shown to be a mediator ofRac1 activity in several morphogenetic activitites during drosophilaembryogenesis, including myogenesis, neural development and dorsalclosure. Thus, these proteins are a part of complex biochemicalsignaling events leading to cell migration and loss of adhesion. Webelieve these proteins are involved in the process and severalcomponents in this pathway are yet to identified. We have a found a genewhich has significant sequence identity to DOCK180. Accordingly, webelieve that DOS is a component that, directly or indirectly, regulatessmall GTPases and actin polymerization. Since DOS genetically exhibits aloss of function in a mouse tumor, we believe that one of the DOSfunctional activities is as a negative regulator of actin polymerizationand cell migration. Additional DOS functional activities are directed totumor invasion and metastasis.

Tissue Expression of DOS by Northern Blots and in situ Hybridization

DOS tissue expression at the RNA level using Northern blots (Clontech,Palo Alto, Calif.) is assessed. These blots are probed with cDNAfragments from the 3′ and 5′ end to identify alternate splicing. In themouse, developmental stages are assessed by Northern analysis. We haveshown by RT-PCR that DOS is ubiquitously expressed. It is not surprisingthat DOS is present in every tissue because it is likely to be involvedin fundamental processes such as, but not limited to, actinpolymerization. DOS-specific, small RNA probes for in situ hybridizationalso are prepared and used to assess mouse developmental stages.

Generating Epitope Tagged Expression Vectors for DOS and DOS Mutants

Epitope tagged, full length construct for eukaryotic expression ispreferably made in pCDNA3.1/V5-His TA TOPO vector (Invitrogen, Carlsbad,Calif.). This vector offers several advantages including versatileexpression driven by CMV promoter and detection of the fusion proteinwith V5 antibody or His antibody. DOS cDNA is amplified in 2 Kbfragments by RT-PCR using PwoI DNA polymerase for low error rates.N-terminal SH3 domain and C-terminal proline rich domain deletionconstructs are made. Detection of fusion proteins on Western blots isoptimized.

Preparation of Antibodies to DOS

Polyclonal antibodies to the DOS protein are made forimmunoprecipitation and immunofluorescence studies by cloning the Cterminus into vector pGEX2T. The C-terminus has no homology to DOCK180and is virtually identical in human and mouse DOS which makes it a goodchoice for antibody production. Antibodies are affinity purified andtested for detection of native DOS in both mouse and human cells.

Expression and Sub-Cellular Localization of DOS Protein in OsteosarcomaCell Line 3081

Studies on DOS protein expression are performed after transfection ofepitope tagged constructs in osteosarcoma cell line 3081. This mousetumor cell line has no endogenous expression of DOS because of DOS genedeletion. After transient transfection of the full length and mutantconstructs in 3081 cell line, its effects on cell growth, proliferationand cell shape are determined using conventional assays. Cells arestimulated by growth factors such as EGF, PDGF, FGF and insulin.Immunofluorescence studies with anti-epitope antibodies are used todetermine DOS sub-cellular localization.

A number of studies have reported that activation of Rho GTPasesrequires membrane translocation. Transfection of wild type DOCK180 inNIH 3T3 cells show its accumulation in the cytoplasm. When DOCK 180 wasfused at the C-terminus with farnesylation signal of ras, DOCK180localized mostly to the membrane. There was a change in shape of thefibroblasts to a pancake morphology which is suggestive of activation ofRac1. In the case of transient transfection studies with DOS, it isconceivable that there is cytoplasmic staining in quiescent cells. Theeffect of growth factor receptors such as EGF, FGF, PDGF and insulinstimulation on cell shape and/or DOS localization is determined. Studieson cell shape and/or DOS localization are done after activation ofintegrin mediated signaling by plating cells on vitonectin orfibronecton and activation of non-integrin mediated cell adhesion byplating cells on polylysine. Farnesylation signal also is fused at the Cterminus of DOS to mimic DOS activation and the effect on cell shapeand/or DOS localization/activation is determined.

Effect(s) of Rho, Rac and CDC42 on DOS

Following the selection of a particular DOS expression phenotype,dominant negative constructs for Rho, Rac and Cdc42 are used incotransfection studies to determine whether specific inhibitors of smallGTPases affect the selected DOS function. Antibodies to DOS are used inimmunofluorescence studies with confocal microscopy to assess DOSassociation with focal adhesion complexes, actin-myosin stress fibers,membrane ruffles, lamellipodia and/or filopodia. These structures arespecific to activation of small GTPases.

Regulation of small GTPases occurs at the level of nucletotide exchangeand is mediated by three distinct proteins families: 1) GEFs or guanineexchange factors which activate GTPases in response to extracellularsignals, 2) GAPs which inhibit GTPases by maintaining it inpredominantly GDP bound form, and 3) GDIs which bind to the GDP boundform and prevents both spontaneous and GEF mediated release of GDP.Thus, there seems to be an exquisite control for the activation of smallGTPases. Co-transfecting DOS with either GEFs or GAPs is performed toassess the role of DOS in GTPase activation.

Protein Association Studies with DOS

Protein association in signal transduction often involves organizationof multi protein complexes via modular components such as SH2, SH3 andPTB domains. SH3 domains bind proline rich peptides and several SH3domains and their cognate proline rich sequences have been established.These interactions are involved not only in protein association butoften target proteins to different sub-cellular destinations. DOCK180has two consensus Crk SH3 binding sites in the C-terminus that arecritical for the formation of the integrin mediated CrkII-DOCK180-Rac1complex. DOCK180 and DOS have no significant sequence homology at theC-terminus. DOS shows sequence homology to 3BP1, a mouse protein thatassociates with Src, Abl and Grb2 via SH3 domains. Therefore, theleading candidates for DOS C-terminal binding are Src, Abl and Grb2.Although not wishing to be bound to any particular theory or mechanism,we believe that DOS has at least 3 functional domains which mediate itsbiological function:

-   -   C-terminus (amino acids 1701 to 1966) putative SH3 binding        sites.    -   N-terminus (amino acids 10 to 100) an SH3 domain.    -   Region of homology to DOCK 180 (amino acids 110 to 1100)        putative small GTPase binding site.

Protein binding studies require cloning the individual domains in GSTfusion vectors. GST pull down assays have been used successfully tostudy the interaction of SH3 domains with proline rich residues. GSTfusion proteins are incubated with cell lysates and associated proteinsresolved by poly acrylamide gel electrophoresis and transferred tonitrocellulose membranes. Bound proteins are identified by Westernblotting with specific antibodies to signaling proteins. Alternately,GST-fusion proteins from Crk, Src, Abl, Nck and Grb2 are incubated withcell lysates expressing epitope tagged full length and C-terminusdeletion DOS constructs. Antibody to the epitope is used to detectbinding of DOS to its cognitive SH3 domain. Also, DOS SH3 domain is usedin such experiments. DOS N terminal SH3 domain may interact with DOSC-terminus leading to both intra and intermolecular interactions.Alternately, the SH3 domain binds to another molecule, thereby,affecting the sub cellular localization of DOS.

DOCK180, when cotransfected with the Rho specific GEF Vav, seems toincrease Rac activity. This is believed to be due to the ability ofDOCK180 to bind to nucleotide free Rac, an intermediate in guanineexchange reaction catalyzed by Vav. Using GST fusion proteins for Rho,Rac and CDC42, the binding for DOS is assessed. Binding of DOS to smallGTPase may require addition of nucleotides GDP or GTP-γ-S. Since, thereare differences in amino acid sequence, it is possible that DOS mayinteract with Rho or CDC42 but not Rac which has shown to be able tobind DOCK180.

The invention is not limited by the proposed mechanisms and/or theoriesdisclosed herein. The above described experiments are based, in part, onthe functional domains present in DOS that are disclosed herein;however, predicted interactions may not be observed experimentally forvarious reasons. Accordingly, additional experiments such as a moreconventional yeast two hybrid approach is used to confirm one or more ofthe DOS functional activities disclosed herein. In this approach,individual domains of DOS are used as baits to clone interactingproteins. The binding of these proteins is further characterized in vivoin mammalian cells.

Physiological Studies on DOS

Experiments designed to address the physiological role of DOS expressionin cells are determined by results from biochemical analysis andidentification of the major signaling pathway. Since DOS was cloned as adeletion in tumor, we believe that loss of function of DOS offerssurvival advantage to tumors. The following experiments directed totumor invasion and angiogenesis assays are performed to assess this DOSfunctional activity. Briefly, osteosarcoma cell line 3081, which has adeletion in DOS is transfected with full length and C terminal mutantDOS. Stable cell lines are generated and Western blots are done toassess the expression of DOS proteins. These cells are injectedsubcutaneously and mice are observed daily and sacrificed one week afterinjection. Tumors are fixed and sectioned and studied for theirinvasiveness. Similarly, angiogenesis is studied by sacrificing mice sixweeks after injection. To study invasion, a G8 myoblast layer is fixedin six well plates. These layers are seeded with stable transfected 3081cells. After one week, cells are observed for invasion by microscopy.Such experiments require optimal controls and are repeated withdifferent isolates of stable transfectants. Following observation of aphenotype in these assays, different molecular components of DOS arefurther tested. In addition, other cell migration experiments areconsidered such as the Transwell migration assay with transfected COS-7cells. This assay has been particularly useful in the identification ofthe Crk/Cas complex as a key switch in the initiation of cell migrationand actin cytoskeleton reorganization. Transwell migration assay withCOS-7 cells is more amenable to experimental manipulation and, thereforeis used to further characterize the molecular events underlying in DOSfunctional activities.

Role of DOS in Human Tumorigenesis

Human cancer cell lines were screened for DOS mutations and missensemutations in the coding sequence of DOS were found in those cell lines.Tables 3 discloses exemplary Mutant DOS molecules. Data show thenucleotide position, and the nature of the nucleotide change in thehuman DOS nucleic acid molecule (SEQ ID NO: 1) and the correspondingamino acid change in the human DOS protein (SEQ ID NO: 2) generated bythe mutations. To illustrate, SEQ ID NO: 5 refers to a guanine toadenine substitution at position 5650 in the human DOS nucleic acidmolecule (SEQ ID NO:1); SEQ ID NO: 6 refers to the corresponding valineto methionine substitution in the human DOS protein (SEQ ID NO:2). TABLE3 DOS mutations in human cancer SEQ Amino SEQ Effect Cell TissueNuclcotide ID Acid ID on Freq. in Line Origin Position Mutation NO:Status Position Mutation NO: Protein normals Mutations U251 CNS 5650 Gto A 5 heterozygous 1884 Val to Met 6 missense 0 of 187 SNB19 CNS 5650 Gto A 7 heterozygous 1884 Val to Met 8 missense 0 of 187 HCT15 colon 260C to T 9 heterozygous 87 Thr to Ile 10 missense 0 of 190 HCT15 colon3325 A to C 11 heterozygous 1109 Lys to Thr 12 missense 0 of 187 HCT15colon 5263 T to C 13 heterozygous 1755 Ser to Pro 14 missense 0 of 190OV1063 ovarian 5153 C to T 15 homozygous 1718 Pro to Leu 16 missense 0of 190 DU145 prostate 5153 C to T 17 homozygous 1718 Pro to Leu 18missense 0 of 190 Polymorphisms HT29 colon 5198 C to G 19 heterozygous1733 Pro to Ala 20 missense 2 of 134 MDA MB 231 breast 5777 C to T 21heterozygous 1926 Ser to Leu 22 missense 4 of 63 HCT116 colon 5750 C toT 23 heterozygous 1917 Pro to Leu 24 missense 2 of 63 common common 5740G to A 25 — 1914 Val to Ile 26 missense nd MDA MB 145 breast 1816 G to C27 heterozygous 606 Glu to Gln 28 missense 7 of 38

Example 2

Introduction

We have found missense mutations in established human tumor cell linesderived from breast, prostate, ovarian and colon cancers. Like other CDMfamily members, DOS binds to the tyrosine phosphorylated adaptorprotein, Crk. DOS harboring a proline mutation identified in humancancer does not bind to the adaptor protein Crk. DOS is unique since itis the only CDM family member that binds to c-Src and rescues thephagocytosis defect seen in ced-5 mutants C elegans. In addition,reconstitution of expression of DOS in the DOS null osteosarcoma cellline, altered cell shape and cells were contact inhibited. Therefore,our data support the hypothesis that DOS is a candidate tumor suppressorgene that appears to function as a bottleneck in cellular pathwaysresponsible for modulation of actin cytoskeleton during morphogenesis,phagocytosis and cancer.

In order to understand how DOS functions in such fundamental cellularpathways we took the opportunity to compare the biological properties ofDOS null osteosarcoma cells with cells reconstituted with either wildtype or mutant DOS identified from human tumor cell lines. Understandingthe protein network centered on DOS will help elucidate its function incell biology, cancer and potentially identify novel therapeutic targetsin oncology.

Background

The study of cancer cells in culture have led to the description of the“transformed phenotype” which alludes, in part, to the alterations inserum and adhesion dependant growth properties of cells. Furthermore, intransformation there were changes in cell morphology and thesetransformed cells exhibited loss of contact inhibition with uncontrolledcell migration. A vast majority of these changes have been attributed toalterations in the actin cytoskeleton. The small GTPases, Rho, Rac andCDC42, are believed to be responsible for the formation of actinmediated assemblies such as stress fibers, focal adhesions, lamellipodiaand filipodia, in both normal and transformed cells. RDA on optimalhuman cancer specimens in our laboratory showed frequent polymorphicdeletions in the human population. Since inbred mouse models of humancancers lack genetic polymorphism, we hypothesized that RDA on mousetumor samples should dramatically enhance the ability to detectpathological deletions in cancer.

Using mouse RDA, a homozygous deletion was identified on mousechromosome 12 in osteosarcoma cell line, 3081. This region is syntenicto a sequenced contig on human chromosome 7q31 and therefore could beused as input data for gene prediction programs. This approach hasenabled us to identify a novel gene, which is referred to as DOS forDeleted in Osteosarcoma. DOS is located on chromosome 7q31near the LOHmarker D7S523 which is frequently lost in advanced ovarian cancer. Thedeletion on mouse chromosome 12 was large but presumably onlyinactivates DOS because the predicted 5′ and 3′ genes, ZNF277 and DLDrespectively, were intact based on Southern blot analysis.

DOS transcript is of 8.5 Kb in size and was universally expressed at lowlevels based on Clontech multiple tissue Northern blot. The DOS mRNAencoded for a large protein of 1966 amino acids (SEQ ID NO: 2)withpredicted size of 225 KD. We have also identified an alternativelyspliced form of DOS, which lacked an exon encoding 38 amino acids (SEQID NO: 32) very close to the C-terminus of the protein. This spliced outregion contained the tyrosine kinase, c-Src SH3 domain binding consensusamino acid sequence PPVPPR. We, refer to the larger protein of 1966amino acids as DOS+SSB (SEQ ID NO: 2) (with Src SH3 domain binding site)and to the smaller 1928 amino acid protein as DOS−SSB (SEQ ID NO: 30)(without Src SH3 domian binding). Using RT-PCR we observed that cellsgrowing in culture express DOS+SSB only (>50 cell types tested).Expression of DOS−SSB can be observed when RT-PCR is performed on RNAderived from whole organs. It is likely that cells express only oneisoform of DOS and the study of differential expression of the twoisoforms in an organ by in situ hybridization helps understanding thefunction of DOS.

DOS is Mutated in Human Cancer

The human DOS gene is located on the locus 7q31. This locus isfrequently involved in LOH in solid tumors of various histologiesincluding breast, prostate renal and colon. As a preliminary screen tosearch for mutations in human cancer, thirty five established cell linesfrom breast, colon, ovarian and renal cancer available through eitherATCC or NCI, were sequenced. A total of five cell lines derived fromovarian, prostate, brain and colon cancer were found to have missensemutations in DOS. The ovarian (OV1063) and prostate (DU145) cancer celllines carry the same homozygous, missense mutation, changing the prolineresidue at amino acid 1718 to leucine (SEQ ID NO: 16 and SEQ ID NO: 18respectively). The colon cancer cell line has a total of three missensemutations (SEQ ID NOs: 9, 11, and 13) in the DOS coding sequence. Inthis limited screen we have not found inactivating mutations in DOS. Themissense mutations observed are unlikely to be polymorphisms since thesechanges were not found in 190 normal individuals. Also, the proline toleucine mutation showed a loss of binding to adaptor protein, Crk. Theseresults support the hypothesis that DOS is a novel tumor suppressorgene.

Cloning of DOS

Using conventional positional cloning techniques, we were able to cloneboth, DOS+SSB and DOS−SSB in pcDNA3.1 under the CMV promoter. Alsoavailable, is the DOS+SSB with the proline to leucine mutation referredto as DOS+SSB P>L found in ovarian cancer cell line (SEQ ID NO: 16) andprostate cancer cell line (SEQ ID NO: 18).

DOS is a Novel Member of CDM Family of Proteins

DOS has sequence homology to four proteins in the database, namely humanDOCK180, DOCK2, Drosophila myoblast city and C. elegans Ced-5. Theseproteins are conserved in sequence and in function. They are believed tobe involved in the initiation of cell migration by triggering actinpolymerization at the cell edge via small GTPase Rac1. Therefore, theseproteins have been shown to have a role in several fundamentalbiological processes such as morphogenesis, cell migration duringdevelopment and engulfment of apoptotic cells.

Based on sequence homology and functional studies, CDM family membersseem to have evolved from the C elegans gene, ced-5. This gene alongwith ced-2 (CrkII) and ced-10 (Rac) were initially identified to play arole in phagocytosis of dying cells during programmed cell death. Asecond defect in pathfinding during the third phase of distal tip cellmigration was later also observed in these ced mutants. Along with DOSthere are two other human genes in the database that are CDM familymembers, and have also evolved from ced-5, namely, DOCK180 and DOCK2.The protein DOCK180 is universally expressed in all cell types exceptfor lymphocytes, while DOCK2 is only expressed in lymphocyte. DOCK180activates Rac leading to membrane ruffles and affects cell morphologyand cell migration. It also rescues the ced-5 distal tip cell migrationdefect without affecting phagocytosis of dead cells. DOCK2 alsoactivates Rac and plays a critical role in T and B cell migration. Incontrast, our data showed that the rescue of ced-5 with DOS+SSB reversedthe defect in phagocytosis of dead cells without affecting distal tipcell migration. DOS+SSB with proline to leucine mutation did not rescueeither phagocytosis or distal tip cell migration suggesting that in thisassay it is a loss of function allele. DOS−SSB, a splice variant alsodid not rescue the Ced 5 phenotype. With these results in mind, one canenvision the possibility that during evolution, Ced-5 has diversifiedinto at least three mammalian genes; DOCK180 and DOCK2, retain theregulation of cell migration function of Ced-5, while DOS retains theregulation of phagocytosis function of Ced-5.

SH3 Domains of Src and Crk Interact with DOS

In transient expression studies, with epitope tagged DOS constructs inboth 293T cell and COS cells, a protein of the expected size is readilydetected after cell lysis with buffers containing detergents, such asRIPA.

After incubating epitope tagged DOS lysates in SDS containing RIPAbuffer with beads carrying GST-Crk-N terminal SH3 domain, DOS+SSB andDOS−SSB was “pulled down”. However, DOS+SSB with proline mutation wasnot “pulled down” suggesting a decreased binding to Crk. Similarly,GST-Src SH3 domain showed efficient binding to DOS+SSB. No binding toDOS−SSB was observed. DOS−SSB does not contain the sequence PPVPPR, theputative Src SH3 domain binding sequence, hence, does not bind to Src.Notably, there was decreased binding of DOS with the proline mutation.The proline rich, C terminus of DOS engages with Src and Crk and theloss of Crk binding is seen in human cancer. Furthermore, no binding toSH3 domain of Abl and Crk-C terminus was observed.

Cell Growth, Morphology and Actin Cytoskeleton Studies

After transient transfection of several established cell lines includingfibroblast cell lines such as NIH3T3 or in 3081, DOS null, osteosarcomacells, we were unable to detect DOS protein expression Therefore, withthe aim to pursue functional studies, DOS null, osteosarcoma cell linewas transfected with DOS constructs and several independent clonesexhibiting stable expression of epitope tagged DOS or DOS mutant(s) wereidentified and selected for further studies. As a control, the parentalcell line 3081 was transfected with vector only.

For cell growth studies, formal cell counts were done. Five thousandcells from two independent clones, representative for each constructwere plated in duplicate on 10 cm tissue culture plates. Cells werecounted daily on a hemocytometer. Growth curves were plotted on asemi-log graph. DOS null, parental tumor cell line grew with rapiddoubling time. Cell growth was significantly retarded with DOS+SSBreconstitution. The cell reconstituted with DOS+SSB P>L had anintermediate phenotype. These data suggest that reconstitution of DOSaltered cell growth when plated on a plastic substrate.

These studies were extended to include growth on soft agar or anchorageindependent growth. Several tumor cells exhibited anchorage independentgrowth which can be reverted by expression of tumor suppressor genes.Parental, DOS null osteosarcoma cells made large colonies as early as 10days after suspension in agar. Expression of DOS in these cellsinhibited growth on soft agar while expression of proline mutation had apartial phenotype. The cell growth data, both on plastic and soft agarsupport the hypothesis that DOS is a tumor suppressor gene and prolinemutation in DOS appears to produce a loss of DOS function.

Stable reconstitution of the DOS null osteosarcoma cell line 3081 withDOS+SSB, changed the shape of the cells from spindle shaped fibroblastlike cells to a polygonal epithelioid appearing cells. These cells arelarge, flat and show increased stress fibers when stained withphalloidin. On the contrary, cells expressing DOS+SSB P>L are smallcells with a decrease in actin containing stress fiber staining. Themorphology of DOS expressing cells when compared to parental cells didnot show increase in lamellipodia which is a marker for increase in Racactivity and has been observed with expression of DOS homologue,DOCK180. The increase in stress fiber does suggest an increase incellular Rho activity. Biochemical studies to elucidate the role ofGTPase activation by DOS were carried out and are described below.

DOS Activates RAP 1

Small GTPases such as Rho, Rac and CDC42 play a critical role in diversecellular functions as cell growth, cell movement and signaltransduction. These proteins shuttle between an inactive GTP bound stateand active GTP bound state. To determine positive and negativeregulators of GTPase activation cascade, biochemical assays with invitro binding selectively to the GTP bound state after cell lysis andcomparing it to the levels of total (GTP and GDP bound form) have beenestablished. Typically, these assays require co-transfection of putativeactivator with construct encoding GTPase in 293T cells. Point mutationsin GTPases, which are dominant-negative and persistently active,respectively, were also used in the assay as positive and negativecontrols.

Both isoforms of DOS, when co-transfected with Rho, Rac and CDC 42 didnot lead to GTPase activation. Other known members of CDM proteinfamily, DOCK2 and DOCK180, are activators of Rac1. Our C elegans datashowed that DOS is distinct from DOCK180 and presumably mediated itsactivity via another cellular GTPase. We turned our attention on Rap1for the following reasons. As described above, all CDM protein familymembers bind adaptor protein Crk. The vast majority of Crk was bound toa protein called C3G which upon being tyrosine phosphorylation activatesGTPase Rap1. There is little information available on Rap1 function.Since, Rap1 was cloned as a suppressor of Ras transformed cells, it wasproposed that Rap1 is an antagonist of Ras and is anti-mitogenic. InDrosophila, Rap1 is extensively studied and is thought to play acritical role in cell-cell adhesion junctions.

Rap1 was co-transfected with DOS in 293T cell and lysates were bound toGST fusion protein of Ra1GDS-RBD domain which has a high affinity forGTP-Rap1. Total lysates were run on SDS-PAGE separately and ratio ofactive GTP-Rap1 to total cellular Rap1 was determined. Rap1 wasactivated by both isoforms of DOS, however, DOS P>L mutation was unableto do so. Persistantly active Rap, Rap63E, and dominant-negative mutantRap, RapN17 were used as controls. Activation of Rap1 by DOS suggeststhat the putative tumor suppressor function of DOS may be mediated byRap via inactivation of the mitogenic MAP kinase pathway and activationof cell-cell junctions.

DOS and Adherens Junctions

The DOS reconstituted osteosarcoma cells were used to study adherensjunctions. These junctions are formed by the interaction of E-cadherinon neighboring cells. The intra-cellular tails of E-cadherin are boundto both a and b catenin. These junctions can be readily visualized usingimmuno-fluorescence when antibodies to beta-catenin are used. The DOSnull osteosarcoma cell lines did not form adherens junctions andexhibited a complete loss of contact inhibition. The DOS reconstitutedcells formed adherens junctions. There was no adherens junctionformation and the cells appeared similar to the parental line when theseosteoparcome cells were reconsitituted with DOS P>L. These observationsvalidated the biochemical data on Rap1 activation by DOS and suggestedthat adherens junctions, a marker for Rap1 activity, can be rescued in acell line that has lost DOS expression during tumorigenesis.

DOS Rescues Tumor Invasion

Although, suppression of growth on soft agar is indicative of tumorsuppressive function, these assays are not always reliable. To addresstumor formation in nude mice, subcutaneous injections of DOS nullosteosarcoma 3081, 3081 reconstituted with either wild type or mutantDOS were performed. Mice were sacrificed 6 weeks after injection. DOSnull and DOS mutant cell lines formed large tumors which invadesurrounding fat, skin and muscle layers. DOS expressing tumors formedsmall, non-invasive, well-circumscribed tumors with surrounding muscle,skin and fat intact.

Our results suggest that DOS has tumor suppressor function. DOS istargeted by mutations in human cancers such as prostate, ovarian, colonand brain. These mutations are missense and are not polymorphisms, atleast, the proline to leucine mutation seen in prostate and ovariancancer cell lines. DOS suppresses growth in soft agar and suppressestumor volume in nude mice. DOS activates Rap1, leading to formation ofcell-cell junctions. Loss of DOS results in disruption of thesejunctions and these junctions can be rescued by reconstitution of DOSexpression. It is likely that loss of adherens junctions lead to celldetachment and subsequent tumor invasion.

We are using RNA interference on DOS in normal cells and studying lossof adherens junction formation. Also, in osteosarcoma 3081 reconstitutedwith DOS, we are disrupting Rap activity and studying cell junctions andtumor formation in mice.

A mouse knockout model is also generated to assess DOS functionalactivities.

All references disclosed herein are incorporated by reference in theirentirety.

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1. An isolated nucleic acid molecule selected from the group consistingof: (a) nucleic acid molecules which hybridize under stringentconditions to a nucleic acid molecule having a nucleotide sequence setforth as SEQ ID NO:1 or SEQ ID NO:3, and which code for a DOS protein,(b) deletions, additions and substitutions of the nucleic acid moleculesof (a), (c) nucleic acid molecules that differ from the nucleic acidmolecules of (a) or (b) in codon sequence due to the degeneracy of thegenetic code, and (d) complements of (a), (b) or (c).
 2. The isolatednucleic acid molecule of claim 1, wherein the isolated nucleic acidmolecule comprises SEQ ID NO:1.
 3. The isolated nucleic acid molecule ofclaim 1, wherein the isolated nucleic acid molecule comprises SEQ IDNO:3.
 4. An isolated nucleic acid molecule selected from the groupconsisting of: (a) a unique fragment of the nucleotide sequence setforth as SEQ ID NO:1 or set forth as SEQ ID NO:3 between 12 and 115nucleotides in length or more, and (b) complements of (a), wherein theunique fragments exclude nucleic acids having nucleotide sequences thatare contained within SEQ ID NO:1 or SEQ ID NO:3, and that are known asof the filing date of this application.
 5. The isolated nucleic acidmolecule of claim 4 wherein the isolated nucleic acid molecule comprisesSEQ ID NO:
 31. 6. An isolated nucleic acid molecule selected from thegroup consisting of: (a) nucleic acid molecules which hybridize understringent conditions to a nucleic acid molecule having a nucleotidesequence selected from the group consisting of SEQ ID NOs:5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, or 29, (b) deletions, additions andsubstitutions of the nucleic acid molecules of (a), (c) nucleic acidmolecules that differ from the nucleic acid molecules of (a) or (b) incodon sequence due to the degeneracy of the genetic code, and (d)complements of (a), (b) or (c).
 7. An expression vector comprising theisolated nucleic acid molecule of claim 1 operably linked to a promoter.8. A host cell transformed or transfected with the expression vector ofclaim
 7. 9. A transgenic non-human animal comprising the expressionvector of claim
 7. 10. A transgenic non-human animal which has reducedexpression of a DOS nucleic acid molecule or of a Mutant DOS nucleicacid molecule.
 11. An isolated protein encoded by the isolated nucleicacid molecule of claim
 1. 12. The isolated protein of claim 11, whereinthe isolated protein comprises of the amino acid sequence of selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, and
 30. 13. The isolated protein of claim 11 whereinthe isolated protein comprises SEQ ID NO:
 32. 14. A binding polypeptidethat selectively binds to the isolated protein of claim
 11. 15-17.(canceled)
 18. A composition comprising: the nucleic acid of claim 1,and a pharmaceutically acceptable carrier.
 19. A composition comprising:the protein encoded by the isolated nucleic acid molecule of claim 1,and a pharmaceutically acceptable carrier.
 20. A composition comprising:the binding polypeptide of claim 14, and a pharmaceutically acceptablecarrier.
 21. A method for making a medicament, comprising: placing anactive agent selected from the group consisting of: (a) the isolatednucleic acid molecules of claim 1, (b) the isolated protein of claim 11,and (c) the binding polypeptides of claim 14, in a pharmaceuticallyacceptable carrier.
 22. The method of claim 21, wherein placingcomprises placing a therapeutically effective amount of the active agentin the pharmaceutically acceptable carrier to form one or more doses.23. A method for diagnosing a disorder characterized by aberrantexpression of a DOS molecule, comprising: detecting in a firstbiological sample obtained from a subject, expression of a DOS moleculeor a Mutant DOS molecule, wherein decreased expression of a DOS moleculeor the increased expression of a Mutant DOS molecule compared to acontrol sample indicates that the subject has a disorder characterizedby aberrant expression of a DOS molecule. 24-25. (canceled)
 26. Themethod of claim 23, wherein the disorder characterized by aberrantexpression of a DOS molecule is selected from the group consisting of: acancer, a tumor, a cytoskeleton disorder, and a cell migration disorder.27-43. (canceled)
 44. A kit for diagnosing a disorder associated withaberrant expression of a DOS molecule, comprising: one or more nucleicacid molecules that hybridize to a DOS nucleic acid molecule or to aMutant DOS nucleic acid molecule under stringent conditions, one or morecontrol agents, and instructions for the use of the nucleic acidmolecules, and agents in the diagnosis of a disorder associated withaberrant expression of a DOS molecule. 45-46. (canceled)
 47. A kit fordiagnosing a DOS tumor in a subject comprising: one or more bindingpolypeptides that selectively bind to a DOS protein or a Mutant DOSprotein, one or more control agents, and instructions for the use of thebinding polypeptides, and agents in the diagnosis of a disorderassociated with aberrant expression of a DOS molecule. 48-50. (canceled)51. A method for treating a subject with a disorder characterized byaberrant expression of a DOS molecule, comprising administering to thesubject an effective amount of a DOS nucleic acid molecule to treat thedisorder.
 52. A method for treating a subject with a disordercharacterized by aberrant expression of a DOS molecule, comprisingadministering to the subject an effective amount of an anti-sensemolecule to a Mutant DOS nucleic acid molecule to treat the disorder.53. (canceled)
 54. A method for treating a subject with a disordercharacterized by aberrant expression of a DOS molecule, comprisingadministering to the subject an effective amount of a DOS protein totreat the disorder.
 55. A method for treating a subject with a disordercharacterized by aberrant expression of a DOS molecule, comprisingadministering to the subject an effective amount of a bindingpolypeptide to a Mutant DOS protein to treat the disorder. 56-58.(canceled)
 59. A method for producing a DOS protein comprising providinga DOS nucleic acid molecule operably linked to a promoter, wherein theDOS nucleic acid molecule encodes the DOS protein or a fragment thereof,expressing the DOS nucleic acid molecule in an expression system, andisolating the DOS protein or a fragment thereof from the expressionsystem.
 60. (canceled)
 61. A method for producing a Mutant DOS proteincomprising providing a Mutant DOS nucleic acid molecule operably linkedto a promoter, wherein the Mutant DOS nucleic acid molecule encodes theMutant DOS protein or a fragment thereof, expressing the Mutant DOSnucleic acid molecule in an expression system, and isolating the MutantDOS protein or a fragment thereof from the expression system. 62.(canceled)